DNA isolation Flashcards Preview

BIOC 311 > DNA isolation > Flashcards

Flashcards in DNA isolation Deck (12)
Loading flashcards...
1
Q

when should a kit be use?

A

– 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

2
Q

what is alkaline lysis sol’n 1?

A

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

3
Q

what is alkaline lysis sol’n 2?

A

aka lysis sol’n; NaOH; SDS– >RT

4
Q

what is alkaline lysis sol’n 3?

A

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

5
Q

alkaline lysis miniprep steps

A
  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
6
Q

when should a kit be used?

A

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

7
Q

options for plasmid quantification?

A

3 main options:

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

8
Q

when is EtBr esitmation used?

A

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

9
Q

why do we do digests?

A

dfod

10
Q

compare the gel buffers

A
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
11
Q

what are some uses for acrylamide gels?

A
  • 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
12
Q

how does heatshock facilitate transformation?

A

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