Editing Plasmid MaxiPrep HIC (section)

==== 9/9/2012 Purify DNA using CaCl2 + PEG or Isopropanol Precipitation ====

DNA from 3 ml culture. + 250 ul P1 with or without RNase, 250 ul P2, 250 ul P3 or N3

+ CaCl2 to 1 M or without CaCl2, Spin, Get supernatant, precipitate with either 10% (final) PEG 8000 or Isopropano. remove liquid, rinse with 200 ul PE, spin brieftly, remove liquid, dry, add 50 ul water. Take 10 ul to load on gel.



[[File:Plasmid PEG ppt 9 9 2012.jpg|RTENOTITLE]]

[[File:Plasmid PEG ppt 9 9 2012 2.jpg|RTENOTITLE]]

8: P1 no RNase, P3 + CaCl<sub>2</sub>, then ppt DNA using 10% PEG

9: P1 no RNase, P3 + CaCl<sub>2</sub>, then ppt DNA using 10% PEG

10: P1 no RNase, P3 + CaCl<sub>2</sub>, then ppt DNA using 10% PEG

11: P1 w RNase, P3, then ppt DNA using 10% PEG

12: P1 w RNase, P3, then ppt DNA using 10% PEG

13: P1 w RNase, N3, then ppt DNA using 10% PEG

14: P1 w RNase, P3, then ppt DNA using isopropanol

15: P1 w RNase, N3, then ppt DNA using isopropanol

16: P1 no RNase, P3+ CaCl<sub>2</sub>, then ppt DNA using isopropanol

17: P1 no RNase, P3+ CaCl<sub>2</sub>, then ppt DNA using isopropanol



{| width="200" cellspacing="1" cellpadding="1" border="1"
|-
| <br/>
| 8<br/>
| 9<br/>
| 10<br/>
| 11<br/>
| 12<br/>
| 13<br/>
| 14<br/>
| 15<br/>
| 16<br/>
| 17<br/>
|-
| OD260 ng/ul<br/>
| 24.7<br/>
| 17.5<br/>
| 17.6<br/>
| 118.7<br/>
| 133.3<br/>
| 102<br/>
| 87.8<br/>
| 975<br/>
| 504<br/>
| 157<br/>
|-
| 260/280<br/>
| 1.82<br/>
| 1.87<br/>
| 1.88<br/>
| 1.9<br/>
| 1.86<br/>
| 1.91<br/>
| 1.99<br/>
| 1.96<br/>
| 1.97<br/>
| 2.04<br/>
|}



Conclusion:

*P1 without RNase + P2 + 1M CaCl2 is as good as P1 with RNase for removing RNA.

*10% PEG is good to precipitate plasmid DNA. Isopropanol is not good.