Editing Plasmid MaxiPrep HIC (section)

=== Butyl 650S or 650M to Purify Plasmid DNA ===



1. Spin down 500 ml cells

2. Add 20 ml of P1 (50 mM Tris-HCl, 10 mM EDTA pH8.0) without RNase.

3. Add 20 ml of P2 (200 mm NaOH, 1% SDS solution), lyse for about 5 min.

4. Add 20 ml of P3 (3 M potassium acetate, pH ~ 5.&nbsp; Dissolve 294.5 g potassium acetate in 500 ml dH2O. Adjust the pH to 5.5 with ~110 ml glacial acetic acid&nbsp; and bring to 1 L with ddH<sub>2</sub>O).

5. Add 15 ml of 5 M CaCl<sub>2. </sub>Final conc is 1 M.

1*(60+X)=5*X; (5X-1X)=60; 4X=60;&nbsp;

5. Put on ice for 10 min, spin at 6000 rpm for 10 min. Filter floating stuff with coffer filter.

<strike>6. Collect supernatant, add isopropanol to final&nbsp;70% (adding 2.1 ml isopropanol per 3 ml of clarified lysate).</strike>

7. Spin.

<strike>8. Wash pellet with 70% ethanol.</strike>

<strike>9. Dry and dissolve DNA in TE buffer (10 mM Tris-HCl, 0.1mM EDTA, pH 8.0.) or water, The DNA solution can be frozen at -20.degree. C. until use</strike>

11. Pack a Butyl 650S or 650M column with a bed volume of <strike>20</strike> 3 to 5 ml <strike>(1.6 cm X 10 cm)</strike>.

12. Equilibrate column with 2 CV of 3 M NaCl <strike>2.2M ammonium sulfate (AS) in 50mM Tris-HCl, 10 mM EDTA (TE) buffer, pH 7.4</strike>.

13. <strike>Add ammonium sulfate to final conc of 2.2 M. and dilute with 2.2 M AS in Tris-EDTA, pH 7.4 to 10 ml.&nbsp;</strike> <strike>Add 5 ml of 3.75 M AS to DNA solution.</strike>&nbsp; Add 75 ml of 5 M NaCl to DNA solution.

14. Load to column at 1 ml/min

15. Wash column with 15&nbsp; <strike>35</strike> ml of 3M NaCl <strike>2.2M AS in TE buffer.</strike>, then 15 ml of 80% Ethanol or PE.

<strike>16. Wash column with 55 ml of 1M AS in TE buffer, pH 7.4,&nbsp; collect two fractions: 10 ml and 45 ml.</strike>

16. Elute DNA with 2 ml then 15&nbsp; <strike>50</strike> ml of&nbsp; <strike>TE or</strike> water.&nbsp; (Alternatively collect 1 ml fractions). Discard the first 2 ml.

17. Measure DNA concentration and 260/280 ratio in each fraction. Combine fractions containing DNA.

18. If purity is not good, purify further using Q XL resin.



9/9/2012

==== Purify DNA using Butyl 650M ====

[[File:Plasmid HIC 9 9 2012.jpg|RTENOTITLE]]

1. Plasmid purified with silica miniprep spin column, using P1 with RNase, and N3

2. Plasmid purified using 650M, add CaCl2 (1 M).

3. Plasmid purified from FT of #2, using silica miniprep spin column

4. Plasmid purified using 650M, add CaCl2 (1 M), did not add NaCl at step 13.

5. Plasmid purified from FT of #4 using silica miniprep spin column.

6. Plasmid purified using 650M, add 60 ml 3M ammonium sulfate at step 5.

7. Plasmid purified with silica miniprep spin column, with P1 without RNase, and P3.

Load 10 ul.

{| width="526" cellspacing="1" cellpadding="1" border="1"
|-
| <br/>
| 1<br/>
| 2<br/>
| 3<br/>
| 4<br/>
| 5<br/>
| 6<br/>
| 7<br/>
|-
| OD260<br/>
| 45.8<br/>
| 64.6<br/>
| 8.9<br/>
| 8.7<br/>
| 3.2<br/>
| 580<br/>
| 157<br/>
|-
| 260/280<br/>
| 1.96<br/>
| 1.85<br/>
| 2.01<br/>
| 1.48<br/>
| -<br/>
| 2.1<br/>
| 2.08<br/>
|}

====  ====

==== 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.