I used the Q5® Site-Directed Mutagenesis Kit (NEB) to perform Site-directed mutagenesis of my plasmid(2886 bp) with an insert of 348 bp and Ampicillin resistant. I followed the protocol of NEB for this particular kit and added 25 ng/ul of plasmid DNA along with other necessary components. But when I transformed them after the mutagenesis I received no colonies on plate even after keeping in incubation for more than 16 hrs. What could be the reason?
There are lots of possible reasons, the key is ruling possible issues in or out.
Did you include any controls? For example, did you spread cells on non-selective media - that will tell you if the cells survived the transformation protocol or not. Or transform a control plasmid like pUC? That can tell you if the efficiency is low.
Off the top of my head I can think of:
wrong temperature or time for heat-shock transformation
"zapped" cells with electroshock = dead cells
competent cells are dead/low efficiency/expired in storage
didn't mix plasmid into cells
incorrect selective media
plasmid ligation didn't work
Have you had this sort of issue before? How about other folks in the lab?
I'm sure you'll be able to get this figured out.
Dear Satarupa Deb Sinha
did you checked the PCR amplification on agarose gel before transformation to see if the PCR was fine?
It is strange that you did not obtain colonies since it is more common to have many colonies but not all with the mutant.
However i reccomend you to evaluate to use PIPE cloning for mutagenesis which do not require any specific KIT but just an high fidelity polimerase as Kapa Hifi and MACH1 cell lines.
Article The Polymerase Incomplete Primer Extension (PIPE) Method App...
Article Combining the polymerase incomplete primer extension method ...
https://www.blogger.com/video.g?token=AD6v5dymTB4mmANTKeTOyzXQq7QSUurTLQFPDsGxi4FqyNDpu3YZutcuN0VfLTfDTPRD8edEIDPD6bJZIe_VWirwCNZHkJ1A34BATcDIzXZEbV1UUtRLR6Nc19tU2_t6-Or5Sag2dwU
https://www.blogger.com/video.g?token=AD6v5dx7hIjOrRfLt70Lph2VJXXgyfu7CVeVUqMIzPNb5ySq3fHJF5QSXLTq93C-8iRHI_fT9_JfcwYQiGCHj0uYBMQzkB7DU8_qi34YSr9ZJzA7OYfHQav5-9eO6Idz5f06bydovl8
best
Manuele
Q5 works quite well for mutagenesis; what is the mutation you want to introduce? juste one base, deletion, insertion... you indicates "dded 25 ng/ul of plasmid DNA" hope it is 25ng in the whole reaction not 25 ng/ul 1/ check the PCR (however frequently the PCR is not very strong because you should not make too much cycles .. and the mutagenesis is nevertheless OK). 2/ make the control included in the kit (if there is some) and check the transformation efficiency with it. Contrary to Manuele Martinelli I considere you should not get many clones (because you should not amplify too much with the PCR (12 o 16 cycles) to avoid unwanted mutation and dpn1 digestion reduced considerably the back ground) but they should be at least 50% mutagenesided (if you do not get one mutant in 3 clones ... improve your PCR (lower the hybridization T°))
I wanted to substitute the MLD region of ssrA gene with GFP's 11th beta strand-54 nucleotides (the gene is cloned in the plasmid pTZ57R/T).
how long is the substitution ? does not look like site directed mutagenesis...
There are certain tweaks to regular protocols for SDM with longer inserts, check out this paper:
Geiser M, Cèbe R, Drewello D, Schmitz R. Integration of PCR fragments at any specific site within cloning vectors without the use of restriction enzymes and DNA ligase. Biotechniques. 2001 Jul;31(1):88-90, 92. doi: 10.2144/01311st05. PMID: 11464525
It worked for me with substituting small insert for full gene so I imagine it will work just fine with your substitution.
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