Unckless Lab Notebook

Pooling mRNA Libraries

Equal-Molar Pooling of mRNA Libraries for Sequencing

Second Set of RNASeq Library Preps from Flies

Preparing Libraries from RNA from D. innubila flies Infected with DiNV Set 2

Second Set of RNASeq Library Preps from Cells

Preparing Libraries from RNA from Dinn-1 Cells Infected with DiNV Set 2

First Set of RNASeq Library Preps from Flies

Preparing Libraries from RNA from D. innubila flies Inefected with DiNV

First Set of RNASeq Library Preps from Cells

Preparing Libraries from RNA from Dinn-1 Cells Inefected with DiNV

Second Set of RNA Extracts from Cells

Extracting RNA from Dinn-1 infected with DiNV for RNASeq 2

Injecting Flies with DiNV for Kent and Gut Dissection 3

Injecting Male and Female D. innubila with DiNV for Kent and Gut Dissection 3

Second Set of RNA Extracts from Flies

Extracting RNA from D.innubila infected with DiNV for RNASeq Set 2

First Set of RNA Extracts from Flies

Extracting RNA from D.innubila infected with DiNV for RNASeq

First Set of RNA Extracts from Cells

Extracting RNA from Dinn-1 infected with DiNV for RNASeq

Injecting Flies with DiNV for Kent and Gut Dissection 2

Injecting Male and Female D. innubila with DiNV for Kent and Gut Dissection 2

Infecting Dinn-1 Cells with DiNV for RNA

Infecting Dinn-1 Cells with P4 DiNV for RNA

Plating Dinn-1 Cells for RNA

Plating Dinn-1 Cells for Infection then RNA

Infecting Flies with DiNV for RNA

Infecting Male D. innubila with 1FFU P4 DiNV For RNA

PCR from All Other MB Injeted Flies

p47, TPI, and CO1 PCRs on all other MB DiNV infected flies

p47 PCR on Viral Load Samples

p47 PCR on samples from flies injected with P4 DiNV to look at viral load

qPCR of Hirt and Puregene Extracted Dinn-1 Cells

qPCR of Hirt and Puregene Extracted Dinn-1 Cells To Compare DiNV DNA, PIF3 and TPI Primers

qPCR of Dinn-1 and Dv-1 Cells Inocculatd with DiNV Cell Primers

PCR of Dinn-1 and Dv-1 Cells Inocculatd with DiNV RPL11 and TPI Primers

DNA Extraction from All Other MB Injeted Flies

DNA Extractions From All Flies From MB DiNV Infections

Another Hirt DNA Extraction a DiNV DNA

Using the Hirt Method To Extract DiNV DNA for Later Comparison with Puregene Extraction

qPCR of Dinn-1 and Dv-1 Cells Inocculatd with DiNV

PCR of Dinn-1 and Dv-1 Cells Inocculatd with DiNV PIF3 and Lambda Primers

PCR of Flies Alive on Day 14 MB Infections

CO1, TPI, and p47 PCR of Flies Alive on Day 14 MB Infections to Check for Virus Presence

Injecting Flies with DiNV for Kent and Gut Dissection

Injecting Male and Female D. innubila with DiNV for Kent and Gut Dissection

Extracting DNA from Flies Alive on Day 14 from MB Injections

Extracting DNA from male D. innubila from Day 14 (last day) of the infection experiment to look for virus presence

DNA Extraction from Dinn-1 and Dv-1 Cells Inocculatd with DiNV

DNA Extraction of Dinn-1 and Dv-1 Cells Infected with 10ul of P4 DiNV

Tenth set of Colonies Screened for DiNV Integration

Screening Colonies from 3/15 Electroporation for DiNV Presence Plate 10

Ninth set of Colonies Screened for DiNV Integration

Screening Colonies from 3/15 Electroporation for DiNV Presence Plate 9

Eighth set of Colonies Screened for DiNV Integration

Screening Colonies from 3/15 Electroporation for DiNV Presence Plate 8

qPCR of Myd88 and Dv-1 Cells Infected with DiNV

qPCR of Myd88 and Dv-1 Cells Infected with DiNV to Check for DiNV Growth

DNA Extracts from Myd88 Cell Infection

DNA Extractions from Myd88 and Dv-1 Cells Infected with DiNV Day 0 and Day 5

Seventh set of Colonies Screened for DiNV Integration

Screening Colonies from 3/15 Electroporation for DiNV Presence Plate 7

Inoculating Dinn-1 and Dv-1 Cells with DiNV

Inoculating Dinn-1 and Dv-1 Cells with DiNV for DiNV Growth qPCR

Sixth set of Colonies Screened for DiNV Integration

Screening Colonies from 3/15 Electroporation for DiNV Presence Plate 6

Fifth set of Colonies Screened for DiNV Integration

Screening Colonies from 3/15 Electroporation for DiNV Presence Plate 5

Plating Dinn-1 and Dv-1 Cells for Infection

Plating 2 24 Well Plates with Dinn-1 and Dv-1 Cells for DiNV Infection and qPCR

Fourth set of Colonies Screened for DiNV Integration

Screening Colonies from 3/15 Electroporation for DiNV Presence Plate 4

Third set of Colonies Screened for DiNV Integration

Screening Colonies from 3/15 Electroporation for DiNV Presence Plate 3

Second set of Colonies Screened for DiNV Integration

Screening Colonies from 3/15 Electroporation for DiNV Presence Plate 2

Transfection of Dinn-1, Myd88, and Dv-1 Cells

Using GFP Plasmid to Transfect Dinn-1, Myd88, and Dv-1 Cells to See Transfection Ability

Inoculating Myd88 Cells with DiNV

Infecting 24 Well Plates of Dv-1 and Myd88 Cells with DiNV to See if Myd88 Cells Grow Virus

First 100 Colonies Screened for DiNV Integration

Screening Colonies from 3/15 Electroporation for DiNV Presence Plate 1

Adding Dv-1 and Myd88 Cells to Transfection Plate and Infection Plate

Adding Dv-1 and Myd88 Cells to the 12 Well Plate for Transfection with GFP Plasmid, and Making 24 Well Plates for Myd88 Infection Assay

More qPCR on Hirt and Exonuclease Treated Samples

Testing Amount of DiNV DNA Ratio Between Hirt and Exonuclease Treated DNA With More qPCR

Female innubila 0.05 FFU DiNV Replicate 2 Infections

Female D. innubila Injections with 0.01, 0.05, and 0.005 FFU DiNV Second Replicate

Electroporation of pSPIN-BAC Cells with DiNV for 3rd Integration Attempt

3rd Attempt at Electroporating pSPIN-BAC Cells with DiNV DNA for DiNV-BAC Integration

Replicate 1 of DNA Extractions on D. innubila with High Dilutions of DiNV and PCRs Second Set

Set 2 of DNA Extracts of Male D. innubila From DiNV Infections from 01/31 and PCRs

Plating Dinn Cells for Transfection

Plating Dinn-1 Cells for Transfection with GFP Plasmid

Replicate 2 of DNA Extractions on D. innubila with High Dilutions of DiNV and PCRs

Set 1 and 2 of DNA Extracts of Male D. innubila From DiNV Infections from 02/21 and PCRs

Electroporation of pSPIN-BAC Cells and DNase Test

Electroporating pSPIN-BAC Cells with DiNV DNA and Treating Resulting Cells With DNase to Remove Outside DNA

Using Middle Band Stock DiNV for Infections Replicate 2

Infecting Male D. innubila with Middle Band DiNV to Check Titering Accuracy Replicate 2

Round 1 of DNA Extractions on D. innubila with High Dilutions of DiNV

Set 1 of DNA Extracts of Male D. innubila From DiNV Infections from 01/31

Using Middle Band Stock DiNV for Infections

Infecting Male D. innubila with Middle Band DiNV to Check Titering Accuracy

Another Hirt DNA Extraction and Exonuclease Treatment For DiNV DNA

Using the Hirt Method and Exonuclease Treatment To Get DiNV DNA for Electroporation

Antibody Stating Dv-1 Cells for Titering

Antibody Staining and Counting Fixed Dv-1 Cells That Had Been Inoculated with 2 Stocks of DiNV

Fixing Dv-1 Cells for Titering

Fixing Dv-1 Cells Inoculated with DiNV For Titering

Making Solutions for Virus Titering

Making Wash and Blocking Solutions for DiNV Titering with Already Plated Dv-1 Cells

Diluting DNA from Electroporation Tests and Redoing PCRs

Dilution DNA to 2ng/ul from Electroporation Tests and Redoing PCRs

Testing mixing of pSPIN-BAC bacteria and DiNV DNA

Testing mixing of pSPIN-BAC bacteria and DiNV DNA at Room Temp without Electroporation

Inoculating Dv-1 Cells for DiNV Titering

Serial Dilutions on Dv-1 Cells with Middle Band and P5 Concentrate DiNV Stocks for Virus Titering

Plating Dv-1 cells for virus titering

Plating 2 48 well plates of Dv-1 cells for DiNV titering

Two sets of qPCR on viral load samples

PIF 3 and TPI qPCR on D. innubila injected with dilutions of DiNV from day 0, 1, 3, and 5 set 1 and 2

Testing DiNV Electroporation and DiNV DNA Retention

Testing DiNV DNA Retention in Electroporated Cells

Dilution Infections of D. innubila with 0.05 FFU DiNV Replicate 2

Male and Female D. innubila Injections with 0.01, 0.05, and 0.005 FFU DiNV, Males Second Replicate

Testing Electrocompetent Cells and DiNV Electroporation

Testing if Cells are Electrocompetent with pTWIN Plasmid and Whether DiNV DNA Gets into Cells

DNA Extract and PCRs on Virus Inoculum

DNA Extraction and PCRs on Passage 4 DiNV Used to Inoculate Cells

Colony PCR on Electroporation 2 Set 2

Second Set of Colony PCR Plates from Electroporation 2 of pSPIN-BAC E. coli with DiNV DNA

Diluting DiNV and Injecting Flies Fourth Replicate

Diluting Titered DiNV Stock to Specific FFU and Injecting in Male and Female D. innubila Replicate 4

Colony PCR on Electroporation 2

First Set of Colony PCR Plates from Electroporation 2 of pSPIN-BAC E. coli with DiNV DNA

Electroporating pSPIN-BAC Bacteria with DiNV Attempt 2

Using the Hirt Extracted and Exonuclease Cleaned up DiNV DNA to Electroporate into pSPIN-BAC E. coli Second Attempt

Plasmid and DNA Extraction from 5 Colonies and PCR

Growth, DNA and Plasmid Extraction, and PCR from 5 Colonies from 1st Electroporation Attempt

DNA Extraction and PCR for Colonies 2 and 13

Growth, DNA Extraction, and p47 PCRs on Colonies 2 and 13 from 1st Electroporation

More Colony PCR on Electroporation Plates

More Colony PCR on Plates from Electroporation of pSPIN-BAC E. coli with DiNV DNA Attempt 1

Dilution Infections of D. innubila with 0.05 FFU DiNV

Male D. innubila Injections with 0.01, 0.05, and 0.005 FFU DiNV

Colony PCR on Electroporation Plates

Trying Colony PCR on Plates from Electroporation of pSPIN-BAC E. coli with DiNV DNA

Electroporating pSPIN-BAC Bacteria with DiNV

Using the Hirt Extracted and Exonuclease Cleaned up DiNV DNA to Electroporate into pSPIN-BAC E. coli

Preparing Electrocompetent Cells

Making the pSPIN-BAC E. coli Electrocompetent

Exonuclease Treatment on Hirt DNA Samples 20-25

Exonuclease Treatment on DNA Intended for Use in Electroporation

Hirt Extraction of DiNV Infected Cells for DNA for Electroporation

Hirt DNA Extraction of Dinn Cells Infected with DiNV for 2 Weeks to Prep DNA to use in Electroporation

Second Exonuclease Treatment on DNA Samples

Second Exonuclease V Treatment Test on Hirt Extracts to Attemp to Remove Linear DNA and 6 Hour Gel

All Day Gel on Exonuclease Treated DNA

Running a 6.5 Hour Gel on the DNA Samples Treated with Exonuclease to Check for HMW DNA

qPCR of Exonuclease Treated DNA

qPCR on DNA Through the Exonuclease Treatment to See if it Enriches DiNV DNA Compared to Cellular DNA

Testing Exonuclease Treatment on DNA Samples

Testing Exonuclease V on Hirt Extracts to Attemp to Remove Linear DNA

Taking the 15 Week Samples for the DiNV Experimental Evolution and DNA Extractions

Taking Samples and Fluid Changing Flasks at the 15 Week Mark for Experimental DiNV Evolution in Dinn Cells (~106 days) and Extracting DNA from 1 50ul Sample Per Replicate...

DNA Extractions from 33 Samples of Dilution Injections Round 5

DNA Extractions from 33 Samples of D. innubila Injected with Dilutions of Passage 4 DiNV and Frozen at Days 0, 1, 3, and 5 Post Injection Round 5

Using Dinn Cells Infected with DiNV Pellet and Clarification Pellet for the Hirt HMW DiNV DNA Extraction

Another Set of Hirt DNA Extractions for DiNV DNA using Pellet of Cells Infected with DiNV and the Clarification Pellet from DiNV Purification

DNA Extractions from 46 Samples of Dilution Injections Round 4

DNA Extractions from 46 Samples of D. innubila Injected with Dilutions of Passage 4 DiNV and Frozen at Days 0, 1, 3, and 5 Post Injection Round 4

DNA Extractions from 46 Samples of Dilution Injections Round 3

DNA Extractions from 46 Samples of D. innubila Injected with Dilutions of Passage 4 DiNV and Frozen at Days 0, 1, 3, and 5 Post Injection Round 3

DNA Extractions from 46 Samples of Dilution Injections Round 2

DNA Extractions from 46 Samples of D. innubila Injected with Dilutions of Passage 4 DiNV and Frozen at Days 0, 1, 3, and 5 Post Injection Round 2

Week 12 Exp Evo Sample

Taking Samples and Fluid Changing Flasks at the 12 Week Mark for Experimental DiNV Evolution in Dinn Cells (~84 days)

DNA Extractions from 46 Samples of Dilution Injections Round 1

DNA Extractions from 46 Samples of D. innubila Injected with Dilutions of Passage 4 DiNV and Frozen at Days 0, 1, 3, and 5 Post Injection Round 1

Preparing pSPIN-BAC for Sequencing

Plasmid Mini-prep and Preparation for Sequencing of pSPIN-BAC

Making Glycerol Stocks from the pSPIN-BAC and Checking Antibiotic Resistance

Picking 3 Colonies to Make Glycerol Stocks for the pSPIN BAC and Checking for Kanamycin and Chloramphenicol Resistance

Making LB Plates and Electorporating pSPIN-BAC

Making Fresh LB Plates and Electroporating the pSPIN-BAC Synthesized by Genewiz into NEB Electrocompetent Cells and Plating on Selective Medium

Injecting DiNV for Viral Load During Infection

Diluting DiNV and Injecting D. innubila to For Measuring Viral Load During Infection

PCR and Prep for Sanger Sequencing Experimental Evolution Samples

Suite of PCR Reactions and Preparation for Sanger Sequencing of Fly Homogenate and Week 9 Experimental Evolution DNA Extracts

Diluting DiNV and Injecting Flies Third Replicate

Diluting Titered DiNV Stock to Specific FFU and Injecting in Male and Female D. innubila Replicate 3

Diluting DiNV and Injecting Flies Second Replicate

Diluting Titered DiNV Stock to Specific FFU and Injecting in Male and Female D. innubila Replicate 2

Overnight HMW Gel on Recent Hirt and Puregene Extracted DNA

Overnight 16 Hour Gel

Taking the 9 Week Samples for the DiNV Experimental Evolution and DNA Extractions

Taking Samples and Fluid Changing Flasks at the 9 Week Mark for Experimental DiNV Evolution in Dinn Cells (~63 days) and Extracting DNA from 1 50ul Sample Per Replicate...

qPCR of D. innubila Dead from Infection and Test Hirt/Puregene DNA Extractions

qPCR to Determine Difference in Viral DNA Yield Between the Hirt and Puregene Extracted Dinn Cells, as Well as Adding in Some D. innubila Adults Who Died from DiNV...

Puregene Extraction, PCRs, and Qubit of Dinn Cells with DiNV

Puregene DNA Extraction of Same Dinn Cells Infected with DiNV That Were Used to Test The Hirt Extraction, PCRs of All Samples, and Qubit

Using Dinn Cells Infected with DiNV for the Hirt HMW DiNV DNA Extraction

Hirt Extractions of Dinn Cells and Concentrated Supernatant Infected with DiNV Round 1

Testing Experimental Evolution Primers on Unextrated Samples

Testing 3 Different Primers from Experimental Evolution Project on Supernatant Samples Before Extraction

DNA Extraction and PCRs of Male and Female 16Cq Injection Replicate 2

DNA Extraction and TPI/p47 PCRs of A Few Samples from Replicate 2 of the Male and Female D. innubila 16 Cq Injections

Taking the 6 Week Samples for the DiNV Experimental Evolution and DNA Extractions

Taking Samples and Fluid Changing Flasks at the 6 Week Mark for Experimental DiNV Evolution in Dinn Cells (~42 days) and Extracting DNA from 1 50ul Sample Per Replicate...

Diluting DiNV and Injecting Flies

Diluting Titered DiNV Stock to Specific FFU and Injecting in Male and Female D. innubila

Designing and Testing Experimental DiNV Evolution Primers

Designing Primers around SNPs Potentially Important for DiNV Adaptation to Cell Culture and Testing the Primers

Planning for a Dilution Series of DiNV Infection

Plan for Diluting DiNV to Certain FFU Concentrations for Injection

Second Attempt at Hirt DNA Extraction from Samples from DiNV Purification

Using the Band 1, Band 2, and Pool of 1 and 2 Harvest Samples From DiNV Purification for Hirt DNA Extraction for Viral DNA

Testing 16Cq Nanoject on Male and Female D. innubila Replicate 3

Test Injections of 16Cq DiNV on Male and Female D. innubila Replicate 3

First Attempt at Hirt DNA Extraction from Samples from DiNV Purification

Using the Clarification Pellet Samples From DiNV Purification for Hirt DNA Extraction for Viral DNA

qPCR for Testing DNA for Fly Infection Experiments

Testing Conditions for qPCR When doing Adult Fly DiNV Infection Experiments

Taking the 3 Week Samples for the DiNV Experimental Evolution

Taking Samples and Fluid Changing Flasks at the 3 Week Mark for Experimental DiNV Evolution in Dinn Cells

DNA Extraction and PCRs of 3 Week Experimental DiNV Evolution Samples

DNA Extraction and PCRs of Samples from Day 0 and 21 Days from DiNV Experimental Evolution in Dinn Cells

DNA Extractions of Infection Experiment Flies for Test qPCR

Extracting DNA from Day0 CCM and DiNV, and Dead from DiNV Infection Flies to Use for an Experiment to Determine Best DNA Concentration for qPCR from Fly Infections

Testing 16Cq Nanoject on Male and Female D. innubila Replicate 2

Test Injections of 16Cq DiNV on Male and Female D. innubila Replicate 2, 20 Injections for Kent, and Some D. melanogaster Injections as Well

qPCR of Poke and Inject Day 0 Flies

qPCR of poked and injected D. innubila with 16Cq DiNV fluid at different dilutions

Testing 16Cq Nanoject on Male and Female D. innubila

Test Injections of 16Cq DiNV on Male and Female D. innubila Replicate 1

Inoculating Dinn Cells for Experimental DiNV Evolution

Incoulating 3 Replicate Dinn Cell Flasks for Monitoring DiNV Evolution over 3 Months

DNA extractions and PCRs on day0 poke or inject flies

Single Fly DNA extracts and TPI and p47 PCRs on day0 D. innubila that either got poked or injected with DiNV

DNA extractions and PCRs on 1st Replicate of 16Cq Nanoject Infections

Single Fly DNA extracts and TPI and p47 PCRs on 1st replicate of 16Cq DiNV Nanoinject infections on D. innubila

DNA Extracts and PCRs of Day -6 of DiNV Experimental Evolution

DNA Extractions and CO1 and p47 PCRs of Day -6 of DiNV Experimental Evolution Samples

Plating Cells for DiNV Experimental Evolution Experiment

Plating Dinn Cells for DiNV 3-4 Month Experimental Evolution Experiment

Fixing and Staining Cells for Virus Titering Test

Fixing and Staining Dinn and Dv-1 Cells That Have a Serial Dilution of DiNV to Test Virus Titering Methods

Testing Nanoject II With DiNV Infections 2nd Replicate

Testing Nanoject II Infections with 16Cq DiNV on D. innubila For a Second Replicate

Inoculating Dinn and Dv-1 Cells for Virus Titering Test

Inoculating 48 Plates of Dinn and Dv-1 Cells with a Serial Dilution of DiNV to Test Virus Titering Methods

Testing Nanoject II With DiNV Infections

Testing Nanoject II Infections with 16Cq DiNV on D. innubila

Testing Needle Shearing on Dinn Cells Another Time

Testing Running Dinn Cells Through Needles to Separate Cell Clumps using 25, 26, and 27 Gauge Needles

Testing Fly Injections with Nanoject II

Testing the Nanoject II Injector to use for Virus Fly Infections

Testing Needle Shearing on Dinn Cells

Testing Running Dinn Cells Through Needles to Separate Cell Clumps

Test qPCR for Fly DNA with Various Dilutions

Testing 1ng, 0.1ng, and 0.02ng of DNA as input for qPCR

pAc5 EGFP Plasmid Extraction

Plasmid Midiprep Extraction of pAc5 EGFP

DNA Extractions and PCRs for Test Sterility Infections

Extracting DNA and PCRs for CO1, TPI, 115, and p47 on Single Flies from the Test Infections for Sterility

DNA Extractions and PCRs of D. innubila Stock at Multiple Ages

Checking for Presence of DiNV in Stock D. innubila Flies from 0-7 Days Post-Emergence with DNA Extractions and PCRs

Test qPCR for Freezing Experiment 1

Doing qPCR for Viral Titre on a Test Subset of Samples From Freezing Experiment 1

Testing D. innubila DiNV Infections for Sterility

Using 16Cq DiNV For Infections with D. innubila im New Protocol to Maximize Sterility

Preparing PCR Products for Sanger Sequencing from Freezing Experiment 1

Re-doing a few PCRs for Freezing Experiment 1 to send some samples off for Sanger Sequencing

p47 Band Strength from Freezing Experiment 1

Analyzing Band Strength in p47 PCRs from Freezing Experiment 1 and Planning Samples to Use for qPCR

Checking Stock D. innubila Genome for DiNV

DNA Extractions and PCRs on Stock D. innubila Genome to Check for DiNV Infection

CO1, TPI, and p47 PCRs for the 1st Freezing Experiment

PCRs for CO1, TPI, and p47 to Test for Virus Presence in 1st Freezing Experiment

Test CO1, TPI, 115, and p47 in Multiplex PCRs

Testing the Ability to Do Multiplex PCRs of CO1 and TPI, and 115 and p47

Last Set of DNA Extracts for Freezing Experiment

31 Single Fly DNA Extractions from Replicate 1 of 16Cq Multi-Day Freezing Experiment

3rd Round of 48 DNA Extracts for Freezing Experiment

3rd Round of 48 Single Fly DNA Extractions from Replicate 1 of 16Cq Multi-Day Freezing Experiment

2nd Round of 48 DNA Extracts for Freezing Experiment

2nd Round of 48 Single Fly DNA Extractions from Replicate 1 of 16Cq Multi-Day Freezing Experiment

1st Round of 48 DNA Extracts for Freezing Experiment

1st Round of 48 Single Fly DNA Extractions from Replicate 1 of 16Cq Multi-Day Freezing Experiment

2nd Replicate 16Cq innubila Infections for Freezing Experiment

Second Infection of D. innubila with 16Cq DiNV For Freezing for Viral Load on Day 0, Day 3, Day 5, and Day 10

Plate 9 Transfection

Second Attempt at pAc5 GFP Plasmid Transfection with DinnDiNV 3.1 and S2 Cells

Counting and Plating S2 Cells for Transfection

Passing Cells and Counting and Plating S2 Cells for Plate 9 Transfection

16Cq innubila Infections for Freezing Experiment

Infection D. innubila with 16Cq DiNV For Freezing for Viral Load on Day 0, Day 3, Day 5, and Day 10

Cell Work 5-23-23

Cell Culture Inventory And Plating 3.1 Cells for Transfection on 5-23

pAc5 Transfection of S2 and DinnDiNV Cells

Using Mirus Bio 2020 Reagent to Transfect pAc5 GFP Expression Plasmid into S2 and DinnDiNV Cells

Plating S2 and Re-plating 3.1 DinnDiNV Cells for Transfection

Counting and Plating S2 Cells and Adding 3.1 Cells to Plate 8 For Transfection

16 Cq D. innubila DiNV Infections

Testing Kent’s 16 Cq DiNV Fluid as and Infection Liquid on D. innubila With More Replicates

Planning DiNV Cloning

Planning for Cloning DiNV With pSPIN By Designing BAC and pSPIN

Cell Culture Work 5/3

Passing Cells and Checking on Plated Cells 05/03

Cell Culture Work and Plating D. innubila Cells 04/27

Fluid Changes, Passing Cells, and More De-Clumping of DinnDiNV 3.0 and 3.1 Cells into Plates

Concentrated DiNV Infections with D. falleni

Testing Concentrated DiNV Fluid with D. falleni Infections

pAc5 and 1544 dsRed Plasmid Midipreps

Midiprep Plasmid Extraction of pAc5-EGFP and pBS-99F8-5’HR-attP»Act5C::dsRed<

16 Cq D. innubila DiNV Infections

Testing Kent’s 16 Cq DiNV Fluid as and Infection Liquid on D. innubila

D. innubila DiNV Infections Round 2

Testing Concentrated DiNV Fluid with D. innubila Infections Round 2

Cell Culture Work and Cell De-clumping

Fluid Changes, Passing Cells, and Trying De-Clumping of DinnDiNV 3.0 and 3.1 Cells

D. innubila DiNV Infections Round 1

Testing Concentrated DiNV Fluid with D. innubila Infections Round 1

qPCR of Myd88 and Dv-1 Cell 12-Well Infection Experiment

Cell and Supernatant qPCR of Myd88 and Dv-1 Cell Samples from 12-Well Plate Infection Experiment

Check PCR of RP49 and Dpt

Checking Myd88 Samples with RP49 and Dpt PCR Primers

qPCR of Concentrated D. innubila Supernatant

qPCR of 115 and Lambda on D. innubila Concentrated and Non-concentrated Supernatant Fluid For Viral Quantification

Cell Culture Inventory 02/28 and Fluid Changes 03/08

Inventory of Cell Culture and Fluid Changes, Fluid Transfers, and Passing Cells

DNA Extractions from Concentrated Viral Fluid

Extracting DNA from Ultrafiltered Viral Fluids

Making Primary Cells from D. innubila Ovaries 20230223

Generating Primary Cells from D. innubila Dissected Ovaries

Testing Ultrafiltration for Viral Concentration

Testing Concentration of D. innubila Primary Fluid Infected with DiNV using Ultrafiltration Centrifugation

TapeStations and Final Size Selection for Wen-Juan's Small RNA Libraries

Post-Size Selection and Clean Up of 1 Small RNA Library, Final Small RNA Library Size Selection and Cleanup, and Final Libraries TapeStations

General PCRs on Myd88 and Dv-1 Cell 12-Well Infection Experiment

115, RP49, RPL11, and Lambda PCRs on Myd88 and Dv-1 Cell Samples from 12-Well Plate Infection Experiment

Making Primary Cells from D. innubila Ovaries

Ringers Solution, D. innubila Dissections, and Primary Cell Generation

Myd88 and Dv-1 Supernatant and Cell DNA Extractions from Feb 8th Infection

Extracting DNA from Cell and Supernatant Fluid from Myd88 and Dv-1 DiNV Test Infections

Generating Fly Extract from D. innubila

Making 8mL of Fly Extract from D. innubila for Ovary Primary Cell Culture

Infecting 12 Well Plates of Myd88 and Dv-1 Cells with DiNV

Using 12 Well Plates of Myd88 and Dv-1 Cells to Infect With DiNV and Take Day 0 Samples

pAc5 Transfection of S2, Mys88, and Dv-1 Cells

Using Mirus Bio 2020 Reagent to Transfect pAc5 GFP Expression Plasmid into S2, Dv-1, and Myd88 Cells

Plating Myd88, S2, and Dv-1 Cells for Transfection

Counting and Plating Myd88, S2, and Dv-1 Cells for pAc5 Transfection

Plating Myd88 and Dv-1 Cells for Another DiNV Infection Test

Counting and Plating Myd88 and Dv-1 Cells to Infect Them With DiNV

Cell Culture Inventory 02/03/23

Inventorying cells during the week of Feb. 3rd 2023

qPCR of DiNV Infected Dv-1 and Myd88 Cells

Doing qPCR on DNA Extracts of Dv-1 and Myd88 Cells, Infected with DiNV and Control

Blue Pippin Size Selection on 5 Small RNA Libraries

Doing Blue Pippin Size Selection on 2-6 of Small RNA Libraries for Wen-Juan, TapeStation, and Purification

Dv-1 and Myd88 Cell and Supernatant DNA Extraction

Extracting DNA from Dv-1 and Myd88 Cells and Supernatant from the DiNV Infection Experiment

Small RNA Library Prep of 6 Samples for Wen-Juan

NEBNext Multiplex Small RNA Library Prep On Six Small RNA Samples For Wen-Juan

Testing DiNV Infection on Myd88 Cells

Using Fluid From Primary D. innubila Cells Infected with DiNV to Test Infection on Myd88 cells

Cell Culture Inventory Jan 4th

Inventory of Primary Cells and Fluid Addition

Cell Culture Inventory Dec. 14th

Inventory of Primary Cells

Testing Reduced Ratios of Transfection Reagent to pAc5 GFP Plasmid in Dv-1 Cells

Testing 0.5-0.75ul 2020 Transfection Reagent Volumes for Increased GFP in Dv-1 Cells

innubila Food and Infection Control Test

Testing the Best Food for innubila Infections and the Best Control for Virus Infections

Testing Different Ratios of Transfection Reagent to pAc5 GFP Plasmid in Dv-1 Cells

Testing Transfection Reagent Volumes for Increased GFP in Dv-1 Cells

Testing pAc5 GFP Transfection on DV-1 Cells

Testing Transfection and pAc5 GFP Expression on DV-1 Cells

P. rettgeri Infections on W1118 Harder Pokes

Trying P. rettgeri Infections Again on W1118 Flies And Poking Harder

Testing pAc5 GFP Transfection on innubila Primaries 2

Testing Cell Transfection Reagents and pAc5 GFP Expression Plasmid on Primary innubila Cells with S2 Cells as a Control

Last 24 CO1 and p47 PCRs on DiNV Test Infections DNA

Doing Final CO1 and p47 PCRs on DNA from Single Fly DNA Extractions and extractions to Test for DiNV Infection

Another Food Test for innubila Infections

Testing Half Instant Food With or Without Mushrooms for innubila Infections

Testing Mirus Bio Transfection Reagents on Primary innubila Cells

Testing Cell Transfection Reagents and pAc5 Plasmid on Primary innubila Cells

Addgene Plasmid pSL1142, New Glycerol Stocks and Plasmid Midiprep

Second Attempt At Creating Glycerol Stocks from pSL1142 from Addgene, and Doing a Plasmid Midiprep to Extract Plasmid DNA

Redo of 81 CO1 and p47 PCRs on DiNV Test Infections DNA

Redoing CO1 and p47 PCRs on DNA from Single Fly DNA Extractions and extractions to Test for DiNV Infection

Second DiNV innubila Infection Test

Second Infection Test on D. innubila with DiNV Positive Fluid from Primary innubila Cells Day 26

Testing Mirus Bio Transfection Reagents on S2 Cells

Testing Cell Transfection Reagents On S2 Cells and 2 GFP Expression Plasmids

81 CO1 and p47 PCRs on DiNV Test Infections DNA

Doing CO1 and p47 PCRs on DNA from Single Fly DNA Extractions and extractions to Test for DiNV Infection

P. rettgeri Infections on Multiple Lines

Trying P. rettgeri Infections Again on Multiple Lines of Melanogaster Flies

Another 48 DNA Extractions on DiNV innubila Test Infections

Another 48 Single Fly DNA Extractions on D. innnubila Flies Infected With DiNV From Test Infections

15 CO1 and p47 PCRs on DiNV Test Infections DNA

Doing CO1 and p47 PCRs on DNA from Single Fly DNA Extractions to Test for DiNV Infection

48 DNA Extractions on DiNV innubila Test Infections

48 Single Fly DNA Extractions on D. innnubila Flies Infected With DiNV From Test Infections

BLASTing Plasmidsaurus pSL1142 Results

Checking Failed Plasmdisaurus Seqencing By BLASTing Reads

Testing Food Vials for D. innubila Infections

Testing 4 Food Vial Types on Control Poked D. innubila and Monitoring Survival

Test Infections on melanogaster and P. rettgeri

Infecting D. melanogaster with P. rettgeri for Infections Trainings

D. innubila Infections with DiNV

Testing Fluids From Primary innubila Cells Infected with DiNV From Kent by Infecting D. innubila Flies with the Fluid

Puregene DNA Extraction

Puregene DNA extraction of Hirt Phenol Chloroform Extracted Samples To Use For qPCR Comparison

Preparing pSL1142, pCS2+8EGFP, and pAc5.1B EGFP for Plasmidsaurus Sequencing

Preparing pSL1142, pCS2+8EGFP, and pAc5.1B EGFP for Plasmidsaurus Sequencing

Addgene Plasmid pSL1142, pCS2+8EGFP, and pAc5.1B EGFP Glycerol Stocks and Plasmid Midiprep

Creating Glycerol Stocks from pSL1142, pCS2+8CeGFP, and pAc5.1B EGFP from Addgene, and Doing a Plasmid Midiprep to Extract Plasmid DNA

More Test innubila and D. melanogaster Infections with Serratia

More training on Infections with Serratia on D. innubila and D. innubila

Hirt/Phenol-Chloroform DiNV Extraction Test 3 and PCRs

Third Test of the Hirt Method for Viral DNA Extraction With Phenol Chloroform Purification on DV-2 Cells Infected with DiNV, and p47 and RPL11 PCRs on Extracted DNA

Test innubila and falleni Infections with Serratia

Training on Infections with Serratia on D. innubila and D. falleni

Cell Culture Checkin 0815

Mini Cell Culture Inventory 20220815

qPCR Comparison of Hirt Extraction and Puregene Extraction

Doing qPCR to Compare DiNV Levels in Hirt Extracted DNA and Puregene Extracted DNA

Hirt/Phenol-Chloroform DiNV Extraction Test 2

Second Test of the Hirt Method for Viral DNA Extraction With Phenol Chloroform Purification on DV-2 Cells Infected with DiNV

Searching for Constitutive Promoters for _D. innubila_

Using D. melanogaster Constitutive Promoters to Search For Promoters for D. innubila

Trial Making D. innubila Fly Extract

Testing Out Making Fly Extract From innubila Flies

PCRs and HMW Gel on Hirt Extraction Test Samples

p47 and RPL11 PCRs and a HMW Gel on the Hirt/Phenol-chloroform Virus Extraction Samples

Hirt/Phenol-Chloroform DiNV Extraction Test 1

Testing the Hirt Method for Viral DNA Extraction With Phenol Chloroform Purification on DV-2 Cells Infected with DiNV

Week of 20220711 Cell Culture Inventory

Cell Culture Inventory on 20220715

Cell Culture Preparation Protocol

Second Updated Protocol For Preparing Drosophila Eggs To Primary Cell Culture

77-2 Test Cas9 Digest

Testing 77kb Guide RNAs 86 and 92 on the 77-2 PCR Product in a Cas9 Test Digestion

p47_q PCR of DiNV Infection Liquid

p47-q PCR on DiNV Primary Day 15 3-23 Used in Infecting Myd88 and W1118 Flies

77-1 Test Cas9 Digest

Testing 77kb Guide RNAs 86 and 92 on the 77-1 PCR Product in a Cas9 Test Digestion

rp49 and p47 PCR on DiNV Infected Myd88 and W1118

Amplifying rp49 (control product) and p47 (DiNV specific) PCR Products on Myd88 and W1118 Flies Infected with DiNV

Cell Culture Work Weeks of 20220516 and 20220523

Cell Culture Fluid Changes and Primary Cell Culture Weeks of 20220516 and 20220523

Cell Culture Inventory on 20220513

Cell Culture Check-in on 20220513

77kb sgRNA Synthesis and Purification

Synthesis of sgRNAs 86 and 92 for the 77kb DiNV Region Using the EnGen® sgRNA Synthesis Kit, S. pyogenes and Purification with the Zymo RNA Clean &...

12 Single Fly DNA Extractions from DiNV Infections

Puregene Single Fly DNA Extractions: 12 Myd88 and W1118 Flies from Infections with DiNV

Infecting Myd88 and W118 Flies with DiNV

Test Infections: Infecting Myd88 and W118 Flies with DiNV and Learning How to Infect Flies

Primary Cell Culture and Inventory 20220502

Cell Culture Inventory and Primary Cell Culture Generation Week of 20220502

Week of 20220418 and 20220425 Cell Culture Work

Cell Culture Care and Primary Cell Culture Generation for Weeks of 20220418 and 20220425

Testing New 130 Region PCR Primers

Test PCRs with New 130kb Region PCR Primers

50ul 77kb PCRs

50ul PCRs for 77kb-DiNV To Make Enough For Cas9 Digestion Assay

Re-visiting Cas9 Region Primers

Rethinking the Primers for Regions of DiNV Needed to Amplify Sequences for Cas9 Digestion Tests

Week of 20220411 Primary Cell Culture and Inventory

Generating Primary Cell Culture and Inventory for Week of 20220411

Cas9 Site for BAC Linearization

Randomly Generating a Set of Cas9 Sites to Use For Potential BAC Linearization - 2 Methods

Cell Culture Week of 20220404

Primary Cell Culture Generation and Cell Culture Maintenance Week of 20220404

Investigating Blunt End Restriction Enzymes

Looking into Blunt End Restriction Enzymes to Use for Linearizing our BAC

Cell Culture Preparation Protocol

Updated Protocol For Preparing Drosophila Eggs To Primary Cell Culture

Cell Culture Week of 20220328

Cell Culture Work for the Week of 20220328: Domeless Primary Cell Generation, Fluid Changes, Secondary Transfer, and Inventory

Testing PCRs for Amplifying DiNV CRISPR/Cas9 Regions

Test Amplifying Primers Designed Here on DiNV to be Used for Cas9 Digestion Tests

Looking for ORI in DiNV

Investigating Where the Origin of Replication is in DiNV/If its in Our Proposed CRISPR Sites

Week of 20220314 Cel Culture Inventory

Cell Culture Inventory on 20220315 of Melanogaster Cells

Week of 2022 02 29 Primary Cell Inventory

Primary Cell Inventory Week of 2/29/22 Check on Flasks and Image

Plasmid Midi Prep on pBeloBAC11

Using Qiagen Plasmid Midi Prep Kit to Extract pBeloBAC11 Using 150mL Overnight Culture

Picking sgRNA Site, and Designing sgRNA Primers and Cas9 Digest Primers

Picking 2 DiNV Regions for CRISPR Sites, Picking 2 sgRNAs From Each

Cell Culture Checkup

Going Through the Cell Culture Flasks Since I Started D. innubila and No Longer Using Trypsin

2nd Try Plasmid Midi Prep on BACs

Using Qiagen Plasmid Midi Prep Kit to Extract pBeloBAC11 and pBACe3.6, Using 25mL Overnight Culture

Considering Using Cas9 Nickases for DiNV BAC CRISPR

Thoughts on Using 2 Cas9 Nickases on to Make the DiNV BAC

Plasmid Midi Prep on BACs

Using Qiagen Plasmid Midi Prep Kit to Extract pBeloBAC11 and pBACe3.6

Expanded CRISPR Guide RNA Planning and Design

Re-Checking Guide RNA Sites in DiNV Genome

Primer Design for DiNV 77,000-78,000bp

Designing Primers for the 77,00-78,00bp Region of the DiNV Genome with Geneious

CRISPR Guide RNA Planning and Design

Using EnGen® sgRNA Synthesis Kit, S. pyogenes (NEB #E3322) and Protocol To Design Oligos Needed for Making sgRNAs for DiNV

Week of 20220124 Cell Culture Preparation - D. innubila

Cell Culture Preparation of D. innubila Eggs

Week of 20220124 Cell Culture Preparation - IMD Null

Cell Culture Preparation of IMD Null Eggs

Testing Other DiNV Primers on 3mLC Gel Extraction DNA

Testing p47_q, 115, Lef 9 and Lef 4 PCR Primers on 3mLC Gel Extracted DNA. These Are Other Primers for DiNV

Week of 20220117 Cell Culture Preparation - SPZ

Cell Culture Preparation of SPZ Eggs

Week of 20220110 Cell Culture Preparation - Testing Plate Types for D. innubila

Using 4 Different Laying Plate Types to See What D. innubila Lays on Best

Week of 20220110 Cell Culture Preparation - Domeless

Cell Culture Preparation of Domeless Eggs

RPL11 and p47 PCRs on 3mLC Gel Extractions

PCR on RPL11 and p47 on the 3mLC Gel Extractions, and Testing the GoTaq

Week of 20220103 Cell Culture Preparation - IMD null

Cell Culture Preparation of IMD Null Eggs

Making BAC Glycerol Stocks

Plating pBeloBAC11 and pBACe3.6 from Agar Stabs, Growing Liquid Cultures, and Making Glycerol Stock

Week of 20220103 Cell Culture Preparation - D. innubila

Cell Culture Preparation of D. innubila Genome Eggs

Overnight Gel and HMW "150kb" Adjusted Procedure Gel Extraction

Another Overnight Gel and Zymoclean Large Fragment DNA Recovery Kit on 3mL-120hr HMW DNA Sample, Using Recommendations From Zymo Representatives

Testing p47 PCR on Gel Extraction Samples in 3 Reaction Conditions

Testing Diluted DNA 10ul Reaction Volume, 20ul Reaction Volume, and Diluted DNA in 20ul Reaction Volume for “3mLB” Gel Extractions on p47 PCR (for DiNV detection)

Getting RPL11 to Amplify Gel Extracted Samples

Week of 20211216 Troubleshooting of PCR After Gel Extraction, Mostly RPL11 (Nuclear Gene)

Week of 20211213 Cell Culture Preparation - 14091 Myd88

Cell Culture Preparation of 14091 Myd88 Eggs

Freezing, Unfreezing, and Passaging Vero Cells

Vero e6 Cells: Making a Frozen Cell Stock, Reconstituting Cells from the Frozen Stock, and Passaging Cells

3mLB-120hr Zymo Gel Extractioned DNA PCR with RPL11 and p47

RPL11 and p47 PCR on Gel Extraction Products from Second Zymoclean Gel Extraction to Check for DiNV in HMW Chunk

Overnight Gel and HMW "150kb" Gel Extraction

Another Overnight Gel and Zymoclean Large Fragment DNA Recovery Kit on 3mL-120hr HMW DNA Sample

3mL-120hr Zymo Gel Extractioned DNA RPL11 PCR and HMW 3mL/2mL p47 and RPL11 PCR

PCR RPL11 (Nuclear Genome Control) on Gel Extraction Products from Zymoclean Gel Extraction and HMW Extracts, As Well As p47 PCR on HMW Extracts to Check...

3mL-120hr Zymo Gel Extraction COI and p47 PCR and Gel

PCR of p47 (DiNV) and COI (control) on Gel Extraction Products from Zymoclean Gel Extraction Determine if Virus DNA is in the HMW Fractions

16 Hour HMW DNA Gel and Zymo Gel Extraction

16 Hour Overnight Gel and Zymoclean Large Fragment DNA Recovery Kit on 3mL-120hr HMW DNA Sample

2 and 3mL HMW Extractions

2mL and 3mL Input into the Genomic Tip Blood & Cell Culture DNA Midi Kit HMW Extraction Kit with 120hr DiNV Infected Dv-1 Cells from Kent

Primary Cell Inventory

Inventory, Check, and Imaging of Primary Cells in the Incubator: How Are They Doing?

120hr Sample Gel Extraction DNA COI and p47 PCR

PCR of p47 (DiNV) and COI (control) on Gel Extraction Products from Last Two - Extractions to Determine if Virus DNA is in the HMW Fractions

15.5 Hour Gel of HMW Samples and Gel Extraction for PCR

Second Attempt at Running a ~16 Hour Gel on HMW DNA Samples, Cutting out a “150kb” Region, and Gel Extracting to Get DNA for PCR

16 Hour Gel of HMW Samples and Gel Extraction

First Attempt at Running a 16 Hour Gel on HMW DNA Samples, Cutting out a “150kb” Region, and Gel Extracting

Week of 20211115 Cell Culture Preparation - 33098 Dicer

Cell Culture Preparation of 33098 Dicer Eggs

Practice Gels for HMW DNA - 16 hrs at 4 Degrees

Testing Running a Gel for HMW DNA using the NEB MidRangePGF Marker, Trying to Find a Way to Separate Out the Ladder, 16 hours at 4 degrees and...

HMW Extraction Attempt 5

3mL Input into the Genomic Tip Blood & Cell Culture DNA Midi Kit HMW Extraction Kit on a 0hr and at 120hr DiNV Infected Dv-1 Cells from Kent

Week of 20211108 Cell Culture Preparation - Domeless

Cell Culture Preparation of Domeless Eggs

Practice Gels for HMW DNA - 15 and 20 Hours

Testing Loading and Running a Gel for HMW DNA using the NEB MidRangePGF Marker, Trying to Find the Optimal Time Running: 15 and 22 Hours

Test Gel for HMW DNA

Practice Loading and Running a Gel for HMW DNA using the NEB MidRangePGF Marker

Week of 20211101 Cell Culture Preparation - SPZ

Cell Culture Preparation of SPZ Eggs

HMW Extraction Attempt 4

Attempting Again the Genomic Tip Blood & Cell Culture DNA Midi Kit HMW Extraction on One Cell Control and One DiNV Positive Cell Culture Samples with a Faster...

Cell Culture Preparation Protocol

Protocol For Preparing Drosophila Eggs To Primary Cell Culture

HMW Extraction Attempt 3

Another Attempt With the Genomic Tip Blood & Cell Culture DNA Midi Kit on One Cell Control and One DiNV Positive Cell Culture Samples

Regular DNA Extraction on Samples for HMW

Using the Puregene Cell and Tissue Kit to Extract DNA from the DiNV Positive and Cell Control Samples to Get a Baseline of Extraction Efficiency

Week of 20211025 Cell Culture Preparation - Dicer 2

Cell Culture Preparation of Dicer 2 Eggs

How to Make Apple Juice Plates

Protocol for Making Apple Juice Plates for Fly Laying for Cell Culture

p47 PCR of HMW DNA Extraction from 20211018/19

DiNV Gene p47 PCR and Gel of HMW Extracts and Supernatant from 1st Successful Extraction Attempt, Two Tries on the PCR

When Yeast Has Overrun Cell Culture

What a Flask Looks Like When Yeast Has Outgrown Everything

HMW DNA Extraction Attempt 2

Genomic Tip Blood & Cell Culture DNA Midi Kit on One Cell Control and One DiNV Positive Cell Culture Samples

Week of 20211018 Cell Culture Preparation - IMD Nulls

Cell Culture Preparation of IMD Null Eggs

Innubila Cell Culture Prep Day 1 and 2

Trying Cell Culture Primary Cell Generation on Drosophila innubila Flies/Eggs on 2021 10 14 and 2021 10 15

Domeless Cell Culture 1 Day 2

Domeless Egg Collection and Cell Culture Preparation Day 2

Cell Control HMW DNA Extraction Attempt 1

Trying High Molecular Weight Extraction of “Cell Control” (non-infected) Cell Culture Sample

Domeless Flies Day 1 Cage Setup, Filtering, and Cell Culture Prep

Cage set up for Domeless fly egg laying, then egg collection and cell culture preparation the day after. Trying using trypsin to break up cell clumps.

Notes on Passing DV1 Cells with Kent

Watching Kent Pass (transfer) DV1 Cells to New Flasks

First Try Collecting Eggs and Preparing Cell Culture on My Own, Myd88 Files

Second day of Myd88 laying and cell culture prep

Myd88 Egg Collection and Cell Culture

Myd88 Egg Collection and Cell Culture Prep with Kent (watching) 2021 10 07

Set up myd88 Fly Cage

Set up Fly Cage With Kent (watching) 2021 10 06

Attempting Whole Fly Cell Culture

Attempting Whole Fly Cell Culture with Kent (watching) 2021 10 05

Plate Yeast on Apple Juice Plates

Prepare Apple Juice Plates with Kent (watching) 2021 10 05

Learning how to make yeast paste for fly laying plates

Making Yeast Paste with Kent (watching) 2021 10 04

Fly Maintenance

Fly Transfer with Kistie (watching) 2021 10 04

Autoclave Prepare Glass Dounce Homogenizer

Autoclave Prepare Glass Dounce Homogenizer (for eggs) with Kent (watching) 2021 10 04