I want to introduce a point mutation (change in one nucleotide) into my gene of interest (DNA binding domain)
I have designed primers as recommended on the Data sheet of the kit :
-Both primers contain the mutation
-The mutation located near the center of the primers
-Primers are 45 bp with melting Temperature greater than or equal 78°c
-%GC 55-60%
But when I requested to design theses primers I didn't mention that I want the primers to be purified
For the mutagenesis reaction (50 microliter)
I'm adding 50 ng of DNA template and 125ng of Forward and Reverse primers
1ul of Dntp mix and 1 ul of PFU HF DNA polymerase ,then perform thermal cycling
95°C 30 sec
95°c 30 sec
55°c 1 min
68°c 1min / kb of plasmid length (my plasmid is 10 Kb I tried once for 10 mins and another time 12 min)
for the cycles I have tried 12 cycle and 18 cycle
Following PCR amplification I digest the sample with DpnI (1ul) ,incubate for 1 hr at 37°c then transform
I used the kit plasmid control and a negative control where :
no colonies in the mutagenized samples and the plasmid control kit
however in the negative control (Vector without mutagenesis , I have colonies ) meaning that the transformation works
why I don't have colonies in the mutagenized samples? what could I change?
Hey Leen Nassar
After initial reading of your method, I noticed you don't perform phosphorylation and ligation of your PCR amplicons. These are necessary steps to add phosphate to the ends of your mutated amplicons and re-circularize them. You can learn more by watching this video: https://www.youtube.com/watch?v=7xFvaI7coyg
I'm also curious why both primers contain the mutation? As far as I know, only one primer must contain the mutation, while the other anneals just beside it (again, see the video to know what I mean).
Hope this helps.
Did you run out a sample of your PCR generated product before (and after) you set up the Dpn1 digest?
If yes, please let us know what you saw.
If no, that's one place to start trouble-shooting.
It's good that you included the vector as a transformation control. That means you can rule out any global issues with the transformation protocol.
Good luck!
Be sure that you have verified your steps according to the company's protocol "QuikChange® Site-Directed Mutagenesis Kit" (www.stratagene.com).
1. The fact that you used two primers is correct, but they really needed to be cleaned. If you haven't already done this, then work with primers you have, however, be prepared that you will have to check more clones to find the correct sequence you want.
2. Be sure to calculate the annealing temperature according to the formula written in this protocol "QuikChange® Site-Directed Mutagenesis Kit" (www.stratagene.com)!!
3. Next, be sure that you have taken a working plasmid isolated from the correct strain (dam+). It is important for DpnI that the working plasmid remains methylated.
4. Another significant note about the work of DpnI: if you have no colonies after all steps you think you did everything right, then try restriction by DpnI for not 1 hour, but for 45–50 minutes.
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