Plasmid-CTAB-Max-Prep
1. Inoculate 500 mL 2xYT or LB/Amp or Kan from 1-5 mL overnight
2. Grow at 37 ºC overnight.
3. Spin cells at 6000 rpm for 10 min at 4ºC using GS3 rotor.
4. Resuspend cells in 8 mL P1.
5. Add 8 mL P2. Stir very gently using a long 5 ml plastic pippette, until homogenous and clear. Put on ice for 10 min.
6. Add 10 mL P3 or S3 (Qiagen), mix, put on ice for 10 min. S3 is the best.
7. Spin 6000 rpm at 4ºC for 10 min.
8. Filter supernatant using autoclaved cheesecloth or coffee filter paper.
9. Add CTAB solution to filtrate at final concentration 0.25% (e.g., add about 1.3 ml to 10 ml filtrate, i.e., 2 ml to 16 ml filtrate), spin at 6000 rpm for 10 min.
10. Resuspend pellet in 1 ml autoclaved 0.65 M NaCl, wash tube with 2 X of 1 mL 0.65 M NaCl (wash 2 times), combine liquid and transfer to a clean 50 ml VWR tube or 15 ml Falcon tube.
---continue to step 11.
Pass supernatant to Qiagen maxi or midiprep spin column---> follow Qiagen maxiprep Plus protocol, label spin column as "G" and "V" for pCMV-Gag-Pol and pCMV-VSVG, respectively, elute DNA with water or EB, do not discard these columns---> analyse DNA by agarose gel and nanodrop.
11. Add 10% Triton X-114 to final concentration of 1% Triton X-114 to the plasmid DNA solution (e.g., add 0.33 ml of 10% Triton X-114 to 3 ml DNA solution). Mix by votexing
12. Incubate on ice for 10 min, mix occasionally.
13. Incubate in a 42°C waterbath for 10 min, mix occasionally.
Note: alternatively, aliquot the solution to 1.5 ml centrifuge tube at this step, and incubate in a heating block at 42 oC.
14. Spin at RT at 6000 rpm for 5 min.
Note: alternatively, spin at max speed using Eppendorf centrifuge if 1.5 ml centrifuge tubes are used.
15. Collect (and combine) upper phase, add 0.43 mL PEG/NaCl/MgCl2 solution per 1 mL liquid. (Add MgCl2 to final concentration of 10 mM).
or add 110 µl of Ca/PEG to 1 ml of liquid.
or add 0.5 ml of PEG-MgCl2 to 1 ml of liquid.
Mix and let incubate at RT for 10 min.
16. Spin at 12000 rpm for 10 min (SA-600 rotor), or maxspeed using Eppendorf centrifuge
17. Wash pellet with 70% ethanol.
18. Spin at 12000 for 1 min.
19. Remove ethanol and air-dry in a biosafety hood.
20. Add 1 ml water to dissolve DNA.
21. Measure DNA concentration and OD260/280 using NanoDrop and run 5 ul on agarose gel to check for the presence of RNA.
Result
[edit]
| Lane |
Sample |
Conc |
260/280 |
| 1 |
pGagPol |
330 ug/ml |
|
| 2 |
V:G, 1:3 | 2ug/ul | |
| 3 |
pGagPol |
1.4 ug/ul |
|
| 4 |
pGagPol, 1:3 |
||
| 5 |
pLentiGPApp, 303 |
1.37 ug/ul |
1.99 |
| 6 |
pLentiGPApp, 303, Precipitate with Ca/PEG |
962.8ug/ml |
1.87 |
| 7 |
pLentiGPApp, 303, Precipitate with Ca/PEG |
237ng/ml |
1.83 |
| 8 |
pLentiGPApp, 303, Precipitate with PEG/NaCl/MgCl2 |
1474ug/ml |
1.89 |
| 9 |
pLentiGPApp, 303, Precipitate with PEG/NaCl/MgCl2 |
762ug/ml |
1.90 |
Lane 1 and 2 Respectively, G and V, the supernatant, which adding BB buffer and centrifuge at 6,000rpm for 15 min, keeps following Qiagen maxiprep Plus protocol .RNA concentration is very high.
Lane 3 and 4: G, the pellet after centrifuge keeps following CTAB-Plasmid-Max-Prep, adding Ca2Cl/PEG. There is the trace of RNA.
Lane 5 and 6: V, the pellet after centrifuge keeps following CTAB-Plasmid-Max-Prep, adding PEG/NaCl/MgCl2. There is the trace of RNA.
So after adding Binding Buffer (BB) and centrifuge, the precipitate includes DNA which we need. Also, using CTAB to purify DNA, the DNA which we get still is not very pure.
Buffers
[edit]TE (Tris/EDTA) [500 mL]
25 mM Trisâ¢HCl pH 8.0 [6.25 mL 2M]
10 mM EDTA [10 mL 0.5M]
Autoclave
Add lysozyme
P3
3.0 M potassium acetate pH 5.5
Dissolve 294.5 g potassium acetate in 500 ml dH2O. Adjust the pH to 5.5 with glacial acetic acid (~110 ml). Adjust the volume to 1 liter with dH2O.
for 500 ml: 147g KOAc (3M)
55 mL glacial HOAc
P3U buffer
3M KAc, 5M CH3COOH, 4M Urea.
PEG/NaCl/MgCl2 solution
30% w/v PEG 8000
1.6M NaCl
25 mM MgCl2.
PEG-MgCl2
40% (w/v) polyethylene glycol (PEG 8000)
30 mM MgCl2
Dissolve 40 g of PEG 8000 in a final volume of 100 ml of 30 mM MgCl2. Sterilize the solution by passing it through a 0.22-µm filter, and store it at room temperature.
Ca/PEG
0.4 M calcium chloride
40% PEG 8000
CTAB/NaCl
2% (w/v) CTAB
0.4 M NaCl
2% (w/v) CTAB
Note: it was reported that RNA can be precipitated with 1.4M CaCl2 (add with P3), then plasmid DNA can be precipiated with 10% polyethylene glycol (PEG 8000), 250 mM NaCl. http://www.ncbi.nlm.nih.gov/pubmed/18571491