DNA isolation

Question 1

when should a kit be use?

Answer 1

– If (time saved * cost of the person’s time) < cost of the kit = use a kit
– If you need large quantities of DNA and don’t have an ultracentrifuge handy = use a kit
– If the DNA needs to be high purity because it is needed for a critical experiment, use a kit
• Like transforming an embryo to make transgenic animals
– If the number of samples is huge (maybe 100+ per day)
use a kit (or a robot)
– If you are screening a small number of samples and the only application is digestion, PCR or sequencing = alkaline lysis is sufficient

Question 2

what is alkaline lysis sol’n 1?

Answer 2

aka resuspension sol’n; 12-12.5 pH glucose, Tris-HCl, and EDTA; –> 4 C

Question 3

what is alkaline lysis sol’n 2?

Answer 3

aka lysis sol’n; NaOH; SDS– >RT

Question 4

what is alkaline lysis sol’n 3?

Answer 4

aka neutralization sol’n; potassium acetate; glacial acetic acid; water; store at 4 C

Question 5

alkaline lysis miniprep steps

Answer 5

1. Pelletcells1minute,maxspeed, aspirate/remove supernatant
2. Resuspend in 100 uL Resuspension solution by vortexing
3. Add200uLLysissolution–inverttube5X
4. Add150uLNeutralizationsolution,invert&put on ice 5 minutes
5. Spin5minutes,recoversupernatant
6. Phenol/chloroformextractionorstraightto alcohol/ice precipitation
   • Yield is ~20-30 ug DNA from 1-2 mL culture

Question 6

when should a kit be used?

Answer 6

If (time saved * cost of the person’s time) < cost of the kit = use a kit
– If you need large quantities of DNA and don’t have an ultracentrifuge handy = use a kit
– If the DNA needs to be high purity because it is needed for a critical experiment, use a kit
• Like transforming an embryo to make transgenic animals
– If the number of samples is huge (maybe 100+ per day)
use a kit (or a robot)
– If you are screening a small number of samples and the only application is digestion, PCR or sequencing = alkaline lysis is sufficient

Question 7

options for plasmid quantification?

Answer 7

3 main options:

– Small-volume spectrophotometry – Large-volume spectrophotometry – EtBr estimation

Question 8

when is EtBr esitmation used?

Answer 8

This is useful for estimation of low quantities of DNA, or samples that are not very pure

Question 9

why do we do digests?

Answer 9

dfod

Question 10

compare the gel buffers

Answer 10

TAE
– Tris, Acetic Acid, EDTA
– Better large band resolution than TBE
– Useful for larger bands, but may require buffer recirculation
TBE
– Tris, Boric Acid, EDTA
– Better small band resolution
– Higher buffering capacity than TAE
– Less heat generation than TAE
– Borate can impair enzymatic reactions if DNA is isolated from the gel

Question 11

what are some uses for acrylamide gels?

Answer 11

• DNA Sequencing
• S1 nuclease assay
• DNA footprinting
• RNAse protection assay
• Recovery of siRNA/miRNA
• Gel Shift (EMSA)
• Determining length of microsatellite repeats • Isolating proper length probes

Question 12

how does heatshock facilitate transformation?

Answer 12

lipids and proteins are released from the outer membrane during heatshock to induce pore formation
– Released dye showed lipid loss
– 2D PAGE showed outer membrane and periplasmic
membranes lost to supernatant
• They also found the heatshock induced depolarization of the inner membrane