Paul.xu at 13:25, 23 June 2013

2013-06-23T13:25:36Z

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Problem: Low yield using CTAB precipitation method. 

== Protocol 1 ==

1. Inoculate 500 mL ~ 1 L 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 25 mL P1 without RNAse A.

5. Add 25 mL P2. Stir very gently using a long 5 ml plastic pippette, until homogenous and clear. Put on ice for 10 min.

6. Add 25 mL P3 (3 M KAc, pH 5),  mix, put on ice for 10 min.

7. Spin 6000 rpm at 4ºC for 10 min.

8. Filter supernatant using autoclaved cheesecloth or coffee filter paper.

 

'''Removal of RNA and endotoxin''' 

9. Add 7.5 ml of 5% CTAB in 50 mM NaCl solution (prewarmed at 37 oC to dissolve precipitated CTAB) to supernatant to final concentration of 0.5% (or 1% for higher yield). Mix.

10. Spin down CTAB-DNA complex at 6000 rpm for 10 min at 25 oC (VWR high performance 50 ml tubes). Remove supernatant, spin again for 30 sec and carefully remove all liquid. 

11. Use 75% ethanol, transfer pellet to microcentrifuge tubes. Spin and remove liquid. 

12. Suspend pellet  DNA solubilization solution (1.2 M NaCl, 1.0 M KAc), put on rotary mixer, transfer solubilized DNA to microcentrifuge tubes, add more DNA solubilization solution until pellet is completely dissolved. 

to make 250 ml DNA solubilization solution solution:

75 ml of 4 M NaCl + 85 ml P3, add water to 250 ml. 

 

13. Add 2 ul of 100 mg/ml RNAse A (from Qiagen), mix and incubate at 37 oC for 15 to 30 min. 

14. Add equal vol of isopropanol (0.7 ml), or 2 vol of 100% ethanol, mix and spin down plasmid DNA at maxi speed (microcentrifuge) for 2 to 15 min, at RT. 

15. Remove isopropanol/ethanol. Wash pellet with ~ 0.5 ml 75% ethanol, spin 1 min at max speed. 

16. Remove ethanol. 

17. Add 0.5 to 1 ml TE or EB to dissolve DNA. Use endotoxin-free TE or EB if used directly for transfection without further purification. 

 

18. Optional: removal of residual RNA on Sepharose CL-4B using a buffer containing 1.5 M (NH4)2SO4 ((20 mM Tris-HCL, 3 mM EDTA, and 1.5 M (NH4)2SO 4 pH 7.2). The use of high salt concentration is beneficial because of the different hydrodynamic size of doubled-stranded pDNA compared with small single-strand content such as RNA and other nucleic acids impurities. Therefore, pDNA eluted in the void volume can be clearly separated from RNA

 

19. Optional: Use a reverse phase POROS R1 50 matrix to concentrate plasmid DNA. The matrix has a dynamic binding capacity is between 5 and 1.5 mg pDNA/mL using a high flow rate (500 cm/h), elute DNA with acetonitrile (10% v/v) or use 10% PEG-8000 to precipate DNA.

 

20.  Polishing: Sephacryl S-1000 media in formulation buffer: 20 mM NaH2PO4–Na2HPO4; 1 mM EDTA; and 30 mM NaCl pH 6.7, or 50 mM Tris-HCl, pH 8.0 with 0.2 M NaCl. 

 

6/18/2013

Gel 1: precipitate plasmid with 1% CTAB (middle lane) and isopropanol (left lane).

5 ul + 15 ul 6X loading dye.

1. Before removal of CTAB:

[[Image:CTAB isopropano plasmid.JPG]]

'''2. Removal of CTAB:'''

To CTAB precipitated plasmid DNA in 0.75 M NaCl solution, add 1/10 vol of 3M NaoAc (P3), and 1/2 vol of isopropanol, mix and spin at maxi speed (13000 rpm) for 2 min, at room temp.

wash once with 75% ethanol, remove ethanol.

dissolve pellet in TE.

C: plasmid from CTAB after purification

I: plasmid from direct isopropano precipitation

[[Image:CTAB isopropanol plasmid2.JPG]]

6/19/2013

0.5% CTAB: 1028ng/ul, 50 ul, OD260/280=1.94

1% CTAB:  845ng/ul, 50 ul, OD260/280=1.91

1% CTAB: 886.6 ng/ul, 50 ul, OD260/280=1.96

[[Image:CTAB isopropanol plasmid3.JPG]]

P: plasmid, R: RNA

Conclusion:

Direct isopropanol precipitation:  ~5.4 mg/ml of DNA/RNA mix, 50 ul from 0.5 ml cleared lysate, OD260/280 ratio is 1.1,  lower plasmid yield, lot of RNA

CTAB precipitation then isopropanol precipitation: ~907ug/ul of DNA/RNA mix, 50 ul from 0.5 ml cleared lysate, OD260/280 ratio is 2.07. DNA peak is good.  High plasmid yield,  much less RNA. This prep could be used for transfection if RNA is removed by gel filtration. 

 

 

[[Category:Protocols|Protocol]]

Plasmid MaxiPrep by CTAB Sephacryl S-1000 SF - Revision history

Paul.xu at 13:25, 23 June 2013