problems with plasmid miniprepplasmid prep
Posted 08 April 2013 - 11:05 AM
I am trying to extract a plasmid from E. coli that has my vector and SOEing insert already ligated in. I am using the alkaline lysis method and here lately I have been extracting nothing but genomic DNA (DNA at the top of the gel near the well) and not plasmid..I am running the plasmid on an agarose gel before restriction digest. I am just recently having problems with this. I isolated a plasmid last November using the same method with little to no problems.
Here is the basic protocol I am using:
1. Grow culture overnight in 5 mL of LB with antibiotic (in this case its ampicillin)
2. centrifuge 1.5 mL for 5 minutes and remove all media
3. Resuspend pellet in 150 uL TE and let sit for 5 minutes
4. Add 300 uL if fresh 10 M NaOH and 10% SDS and 3 uL of RNase, mix by inverting 6 times, and let sit for 3 min
5. Add 225 uL Pottasium acetate, and mix by inverting 6 times
6. Centrifuge for 10 minutes
7. Transfer supernatant to new tube and extract with an equal volume of choroform, phenol, isoamyl alcohol
8. Vortex and centrifuge for 5 minutes
9. Transfer supernatant to a new tube
10. Add 2 volumes of cold 95% EtOH and leave on ice for 25 minutes
11.Centrifuge for 5 minutes
12. Remove supernatan and drain tubes on a paper towel by propping them up on a sharpie to allow the EtOH to drain off
13. add 1 mL cold 80% EtOH, and centrifuge for 5 minutes
14. Remove supernatant, drain well nad let dry for 10 minutes
15. Resuspend in 50 uL of TE..
That's what I did last November also when I was not having any problems...and now I am isolating genomic DNA. Any suggestions?
- Teevesuicaree likes this
Posted 08 April 2013 - 02:34 PM
Posted 10 April 2013 - 06:27 PM
Posted 11 April 2013 - 12:51 PM
Make sure that your NaOH solution is fresh, it readily absorbs CO2 from the air which alters the solution.
Check to make sure that your acetate is prepared properly - too little salt will stop the DNA from precipitating well, especially smaller bits.
Try adding the acetate pretty much immediately after mixing with the lysis solution, I tend to find that the shorter the incubation here, the better my minipreps are.
Posted 11 April 2013 - 06:38 PM
Posted 15 April 2013 - 12:26 PM