Hello Maria,

Can you provide more information on the primer design (sequences) vs wild type sequence?

And did you check for correct sized plasmid amplification by agarose electrophoresis after PCR?

My guess would be that the mutagenesis somehow did not work out and you just obtained the small fraction of unmodified, not dpnI digested parental plasmid for transformation then

How many colonies did you send for sequencing?

Best,

Pascal

It seems that your parental plasmid was not digested with DpnI and the pcr didn't occur. After you have DpnI digested the pcr product, run a few ul on agarose gel. You should get a band for the intact mutagenized plasmid (which won't be digested) and a faint smear below indicating digestion of your parental plasmid. Your original plasmid got transformed and produced colonies. If your problem persists even after solving the DpnI digestion, consider changing the primers.

Hii !!

I just have few queries:

1- what is the incubation period and temperature of Dpn digestion which you are performing.

2-What is the concentration of template you are using?

3-and what is the length of overlapping regions in your mutagenic primers

It seems like the Dpn digestion is not working properly

Apart from this method of SDM you can try the following method

You can use overlap extension method for the mutagenesis. Its a full proof method in which you need to design 2 set of primers for your gene of interest.

The 1st set will be Forward primer with the restriction site and the Reverse primer will be the one, which is made for the mutagenic site

The 2nd set of primers should be Reverse from the 3' End and the forward from the mutagenic site .

(Note: The mutagenic primer should be somewhere overlapped)

So You have to perform total 3 PCR Reactions

1- using your template with F-Normal and R-Mutagenic primer

(here you will get your half amplification with the flanking region)

2-Using your F-Mutagenic and R-Normal primer

(here also you will get next half of your gene)

3-Using The gene products of both PCR 1 and 2 as a template with F and R normal primers.

Then just perform the restriction digestion and ligate it in your plasmid

Dear Maria

of course to provide you reilable answer we need to know more detail about primer sequence and condition that you used in the PCR reaction.

However just a question:

how much dna template did you used for the PCR? and did you saw an amplified band corresponding to the amplified vector before dpni digestion?

Because it seems that you used too much dna template, because dpni is not enough in case the template is a lot.

Generally i'm using maximun 0,1ng of vector for a 50ul PCR. What about you?

Unfortunatelly, the PCR colony screening in case of single point mutagenesis is not able to distinguish beetween the original and mutated forms, you can avoid it and send directly to sequence your mini kit.

ciao

Manuele

Dear all,

Thank you for your suggestions.

I am working on a human protein and mutagenising a particular region that acts as a linker between two domains to shorten it. The colony PCR has shown that all screened colonies have the insert of that gene and a smear which demonstrated successful digestion of template methylated DNA by dpn1. By logic, after mutagenesis, KLD and sequencing there must only be present the mutated sequence, but sequencing has shown that no mutation occurred.

For mutagenesis I followed the Q5 PCR protocol: 1 uL of Template, 12.5 uL Q5 Hot start high fideity 2X mastermix, 1.25 uL 10 uM primers (each of fwd and rev), MilliQ H20: 9 uL. PCR: initial denaturation at 98C for 30 sec, 30 cycles of: denaturation (98C, 10 sec), annealing (63C, 30 sec), extension (72 C, 4 min) and final extension (72 C, 2 min). Gel has shown that Q5 mutagenesis was performed successfully.

For KLD, NEB protocol was followed: 1 uL template (from the Q5 PCR), 5 uL of 2X KLD reaction buffer, 1 uL of 10X KLD enzyme mix and 3 uL of MilliQ water. The mix was incubated at room temperature for about 5 minutes, then added to DH5a cells and put on ice to incubate for 30 min. Heat shock was performed for 45 seconds at 42C. Bacteria were incubated on ice again for 5 min. 300 uL of LB was added to bacteria, which were then grown in orbital shaker for 1 hr at 225 rpm at 37C. Bacteria were grown on Amp100 agar plates (100 ug/mL Ampicillin sodium salt) overnight at 37C. Next day separate colonies were propagated for 16 hours in 10 mL LB+ 10 uL of Amp100.

This procedure has successfully worked for one mutagenising primer and I am looking forward to check whether other primers have worked. Otherwise, the simplest troubleshooting is to screen more colonies.

What was the concentration of template used for SDM pcr?

And the concentration of DNA used for dpn digestion?

It seems you have used more DNA so you need to increase the duration of dpn digestion

I would suggest to increase the duration To 2 hours And then check.

On NEB site you can see the enzyme units for a fixed amount of DNA, you can also put reaction according to that.

All the best!!