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problems with plasmid miniprep

plasmid prep

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8 replies to this topic

#1 shawn6022

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Posted 08 April 2013 - 11:05 AM

Hey everyone,

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?

#2 bob1

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Posted 08 April 2013 - 01:16 PM

Usually when doing alkaline lysis the concentration of NaOH is 0.2 M and the concentration of SDS is 1% - the NaOH will be damaging the plasmid DNA.

#3 phage434

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Posted 08 April 2013 - 01:48 PM

Also, 2 volumes of EtOH is usually not sufficient for precipitation. I'd recommend 2.5 volumes.

#4 shawn6022

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Posted 08 April 2013 - 02:34 PM

Thanks. i will try the lower molar NaOH and use 1% SDS instead of 10%. I usually use 2.5 volumes EtOH but I was looking at a different protocol when I typed this out. I hope this works...I manage to get a good amount of genomic DNA when doing this...do you think that the higher molar concentration of NaOH is causing that?

#5 Ahrenhase

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Posted 09 April 2013 - 12:14 PM

Most likely. The genomic DNA should be pulled down with the flocculant after the addition of the potassium acetate. You could be shearing it into pieces at those concentrations of NaOH.

#6 shawn6022

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Posted 10 April 2013 - 06:27 PM

Ok, after doing some calculations the final concentration of the NaOH is 0.2 M. I make the solution with 4.4 mL ddH2O, 500 uL 10 % SDS, and 100 uL 10 M NaOH...the final concentration is 1% SDS and 0.2 M NaOH...

#7 bob1

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Posted 11 April 2013 - 12:51 PM

Right - that's a lot better, you should always work in final concentrations if you want to discuss this sort of thing - otherwise to us it looks like you made a serious mistake as we only know what you tell us...

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.

#8 shawn6022

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Posted 11 April 2013 - 06:38 PM

Thanks and I apologize for the confusion. I will remake the NaOH tomorrow and try what you said about adding the acetate right after the lysis step. I've noticed that different protocols have various times to keep the cells in the lysis solution before adding the acetate..I will try it and let you know how it goes...once again I apologize for the confusion..

#9 shawn6022

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Posted 15 April 2013 - 12:26 PM

Hey, I just wanted to let you know that your method of adding the acetate quickly after the lysis step worked. I was able to isolate a beautiful band of both the insert and the vector after the restriction digest. Thanks again!




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