Puregene DNA Extraction

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

Using the same samples from the previous Hirt extraction, however I used the same volume as input for each extraction this time.

Steps

Thawed samples on ice
Transferred 400ul of each sample to new 1.5mL tubes
Centrifuged tubes for 30 seconds at 13,000 rpm
Visible cell pellet after this, however the pellets in 1.5-2 and 0.75 samples were smaller than 0.5-C sample. This means that the densities of cells in each of these samples are different
Removed the supernatant
Added 300ul cell lysis solution to each sample and pipetted to mix/break up the pellet
Made diluted RNase A (need 4mg/mL)
- 24ul molec grade water
- 1ul 100mg/mL RNase A
Added 1.5ul diluted RNase A
Vortexed and spun down samples
Incubated samples at 37 degrees C for 40 minutes
After incubation, placed tubes on ice for 1 minute to chill
Added 100ul of protein precipitation solution to each tube
Vortexed tubes for 10 seconds
Placed tubes on ice for 5 minutes (tubes got cloudy)
Centrifuged tubes for 3 minutes at 14,000rpm
Visible white pellet after this
Made 3 new tubes with 300ul of isopropanol in each
Moved ~330ul of supernatant to the new isopropanol tubes
Inverted tubes ~50 times to mix
Centrifuged tubes at 14,000rpm for 5 minutes
- The only tube with a visible pellet was the 0.5-C sample
Removed supernatant as best as possible
Added 300ul of 70% ethanol to each tube
Centrifuged tubes at 14,000rpm for 1 minute
- Again hard to see if there was a pellet
Removed supernatant as best as possible
Inverted tubes on a kimwipe for at least 30 minutes to dry
Added 20ul of 10mM tris HCl
Let DNA sit overnight on the bench to resuspend

202200911 Qubit

These are low yields, but this may be because the input volume was small. These are still fine to use for qPCR however.