Second Set of Colony PCR Plates from Electroporation 2 of pSPIN-BAC E. coli with DiNV DNA
I again decided to pick 5 more colonies from each DiNV plate from electroporation 2. The plates had been in the 4C since being grown.
I prepared 50 strip tubes with 5ul of molecular grade water. I also prepared 2 LB plates with chloramphenicol (20ul of 50% stock chlor was spread on each plate and let soak in and warm for ~30 min before use) to keep the colonies on. The plates had a grid layout with 25 spots per plate:
For each colony of the DiNV plates I picked a colony with a new pipette tip, dipped it on the plate square, and then dipped and spun it in the strip tube for that colony. This way I could keep growing each colony. The plates were left to grow overnight and the put in the fridge to store.
Colonies 57-81 were from the DiNV-1 plates. Colonies 82-106 were from the DiNV-2 plates. All information about the colonies can be found here.
All colonies were then run through 3 PCR primers separately and ran out on gels and products scored. PCR followed the general PCR protocol.
Mastermix volumes are shown here:
| Reagent | p47 | 16S | lef9 |
|---|---|---|---|
| GoTaq | 275ul | 275ul | 275ul |
| F primer | 13.75ul | 13.75ul | 13.75ul |
| R primer | 13.75ul | 13.75ul | 13.75ul |
| molecular grade water | 192.5ul | 192.5ul | 192.5ul |
4 separate gels were run because of issues with staining on double laned gels:
