I am trying to mutate a 17 kb plasmid (pTarget) using overlapping primers. I get a clean amplification of > 10 kb. But when I try to transform it, either the colonies turn blue or their are no colonies. ( Phusion DNA pol used for amplification,Dpn I for digestion XL1 blue, Dh5a, HB101 cells used for transformation).
Is their any other way to check the mutagenesis pcc reaction before transformation?
Site-directed mutagenesis with overlapping primers works well but it doesn't do miracles. It works routinely with 3-5 kb plasmids, and you have to sequence 1-2 candidates to be sure and also to check for other mutations that may occur elsewhere. If a plasmid is bigger than 10kb, I always isolate a smaller section by assembly PCR, typically using the same overlapping mutagenesis primers but with a sense primer further upstream and an antisense primer further downstream and gel-purify a sticky ended fragment containing the point-mutagenised region for re-ligation into the large plasmid. Then I only have to sequence that insert. If you do this well (use an empty vector that does not have your wild type sequence in it already), 100% of the recombinants have the mutation. This is an old technique but it works very well.
You can check be increasing the duration of dpn1 digestion for atlaest 3 hrs ( by adding 1ul of enzyme / 50 ul of pcr reaction)
i didn't understand ur doing well but could u please check the mutagenesis program (elongation time must be 34 min for 17 kb plasmid) and after PCR reaction has been done take 5 µl for testing on gel, incubate the rest by 37 C for a hour with 1µl Dpn1.
use 1% agarose gel.
Hi..
u can try with stratagene pfu turbo and dpn (quik change XL mutagenesis kit). It works miracle for me...never failed actually...
I expect phusion to work gud too, but i hv never used it.
ur primers should be of high Tm (>78), 34-40 min extension per cycle
also final extension shud be increased...do not put for more than 15 cycles
u can also add new enzyme after half time...efficiency goes up!!
dpn shud be of 3 hrs for sure
check ur comp cells..they shud be of very high efficiency...
u can also precipitate ur final pcr product with Na-acetate+ethanol...help cleaning the sample...
best of luck!!
@ Jürgen Denecke: Sir, I was trying to clone a 12 kb RNA, I was unsuccessful amplifying the whole RNA. So I made smaller clones and assembled by asymmetric overlap extension pcr and adding some restriction sites.
The biggest fragment that we could assemble by Overlap extension pcr is ~ 8 KB. Since the fragment size is very big It is very difficult to ligate such products in a vector and clone it.
@ Almas Shamaila I believe Dpn I works fine. Otherwise i would have got some colonies when I transformed my template plasmid( methylated) in MrcBC negative cells.
@MoHaMaD ToUtOuNjI and Andaleeb Sajid : I have a clean amplification of with Mutagenesis PCR.
The parent plasmid was isolated from White colony. Now after Mutagenesis PCR Colonies turn blue without Insert.
I do Sod.acetate precipitation and check in gel before transforming it.
We have had relative success mutating large plasmids by SDM. Enzyme and long extension time are key. Check our tutorial here: http://www.primergenesis.com/vali1.php
Hope this helps.
Bruno