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How realistic would it be to amplify a 15Kb plasmid with no errors?
We are currently trying to amplify a plasmid needed for transfection. The problem is that the yield from ecoli amplification is poor since the plasmid is low copy. We have tried chloramphenicol in our culture and it helped somewhat but still not close to what we need. We want to try the PCR route, but I have read that it can be quite tricky. Any suggestions on how to go about this are appreciated.
Hello Mirella,
I think it's doable. I amplified plasmids in the past by PCR and it works pretty okay. They main tips are:
-You will need to use a proof-reading enzyme to minimise the errors and long extension periods for that (10-15 mins depending on the enzymes). If I remember well, I was using Phusion™ High-Fidelity DNA Polymerase (from Thermofisher).
-For the primers, I designed a Forward primer with a Tm of 55-60C and 19-22 bp. The Reverse primer was the reverse complementary of the Forward primer.
-30-35 cycles should be sufficient.
-You will need to sequence the plasmid once you have amplified it.
Give it a go, it is not very difficult to amplify it. Let me know if you have any questions (and if it works! :P). Check this protocol: doi:10.1101/pdb.prot086827. It's the one I was following previously.
All the best,
Roberto
I've used Takara Bio CloneAmp HiFi PCR Premix to amplify plasmids 11 kb in size with no errors for in-fusion cloning, so I'm fairly confident it could work with your plasmid.
I don't think it's a problem. If you really worry about it, you can do a plasmid sequencing after plasmid extraction.
Alternatively, you say it's a low-copy number plasmid. Have you tried other media besides normal LB broth? Maybe you can try 2xYT and terrific broth to see if you can get a higher yield from E. coli.
Side Hu, I may actually try that instead. Have you tried both, if so which worked better?
Mirella, recently I have cloned a gene onto pDEST15, which might be a low-copy number plasmid. I extracted the plasmid but only obtained very little plasmid. Yesterday I used 2xYT for expression (using the same vector), and I got quite a lot of cell pellets. My colleague also confirmed that 2xYT will increase the plasmid yield. But to be honest， I haven't yet tried Terrific Broth but searched for this thing. It is a nutrient-rich medium, even richer than 2xYT. So I think it's worth trying.
Genei XT-20 PCR system can be useful.
https://geneilabs.com/product/xt-20-pcr-system-250-u-3-u-l-enzyme-1vial-10x-assay-buffer-20a-1-vial-10x-assay-buffer-20b-1-vial/
To amplified your plasmid : make the culture to 32°c with 12,5 ug/ml chloramphenicol. Strech your bacteria and pick one colonie to start your 400 ml culture.
For the extraction resuspend in 30 ml of 50mMTris/HCL ph8 10mM EDTA 100ug/ml RnaseA, then pour 30 ml of cold 200mM NaoH (fresh) 1% SDS. Mix gently until you obtain flakes. Leave on ice 2mn. Spin 30mn 12000g. 4°c with angle fix rotor. With the supernatant fill 4tubes (50ml) with 20ml/tube
Add 14ml propanol-2 (0,7vol). Mix several time, leave on 10mn. Spin again
30mn 12000g. 4°c. Wash the pellets with 5ml EtoH70%, spin again. After remove alcool and dry at RT. Dissolve the DNA pellet in 500ul TE/RNASE 20ug/ml, wait 5mn at RT. Precipitation : add 50ul (1/10 Vt) of 3M NaAc pH5,2 and 390ul propanol-2, mix gently until you see a medusa . Pick up with a tip. Wash with ETOH 70%, dry and dissolve in TE. This 2° precipitation allow to separate RNA from plasmid
I use tRNA as a carrier in the first step. With a kit you loose a lot of plasmid that stick to the membrane
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