Merlin Maxipreps (3/3)

Chia Jin Ngee mcblab47 at leonis.nus.sg
Fri Feb 18 03:05:09 EST 1994


Merlin Maxipreps
Reagents needed:
Merlin Preps Reagents (see: Merlin Preps Reagents)

Maxiprep  protocol

1. Grow a 250 - 500 ml culture O/N. Use rich media or amplify plasmids. 

2. Spin the 250 ml O/N culture using a GSA rotor at 7.5 k RPM. 

3. Perform a boiling prep and propanol precipitate the DNA/RNA. 

4. Resuspend in 4 ml of TE buffer. 

5. Aliquot 10 ml of MPS4 into a 50 ml Falcon tube and 40 ml of MPS5 into
it. 

6. Add the 4 ml DNA solution from step 4 to the solution from step 5. Mix
on a shaker platform gently for 30 min. 

7. Shake the slurry well and load it onto a Econo-Pac Column connected to
a vacuum line. Allow the resin bed to form under vacuum. 

8. After the column is sucked dry, add 20 ml of PS5 and wash the column
thru suction. 

9. Allow the suction to continue for 5 min after the column is dry to
allow removal of ethanol. 

10. Disconnect the line and add 5 ml of 70-80C TE buffer to the column.
Place the column in a 50 ml Falcon tube and spin at 2500 RPM in a Sorvall
RT6000 for 5 min at room temperature. Use a swing bucket rotor for this. 

11. Transfer the eluate from the 50 ml tube to a 15 ml Falcon tube. Spin
the column again under the same conditions to collect residual eluate.
Transfer the eluate from the second spin to the 14 ml Falcon tube. 

12. Add 0.1 vol of 3 M sodium acetate and an equal vol of isopropanol.
Vortex and freeze at -80C for 30 min. 

13. Spin on a Sorval RT6000 for 15 min, 4C to pellet DNA. 

15. Rinse pellet twice with 70% ethanol. Dry briefly in Speed Vac.
Dissolve in 500 ml of TE buffer. 

16. Transfer to microfuge tube. Add 1 ml of RNAse A and incubate at 37C
for 30 min. 

17. Phenol/chloroform extract the DNA solution and reprecipitate it with
ethanol. Rinse with 70% ethanol and dry briefly as before. 

18. Dissolve in 500 ml of TE buffer. Quantitate with A260 readings. 

--
Jin Ngee, Chia			Chemical Carcinogenesis Laboratory
(Genie, the OligoMan)		Institute of Molecular and Cell Biology
mcblab47 at leonis.nus.sg		National University of Singapore
Tel: (065)772-3797		Kent Ridge Crescent
Fax: (065)779-1117		Singapore 0511
"I'm Mr Chia not Ms Chia and I know Genie is a girl's name"-Jin Ngee, Chia



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