I have been trying to do a site directed mutagenesis for single aminoacide change, using QuickChange II Site Directed Mutagenesis Kit.
I have designed my primers using QuickChange Tool. The Tm of the primer is 86 celcius degrees and GC content is %62.5.
I made the following PCR reaction:
2,5ul 10x reaction buffer
25ng DNA template
125ng forward primer
125ng reverse primer
0,5ul 10mM dNTP mix
0.75 ul DMSO
0.5ul Pfu ultra polymerase
dH20
Total reaction volume: 25ul
I then set PCR machine at these conditions and cycled 16 times.:
95 C, 30 secs
95 C, 30 secs
55 C, 1 min
68 C, 7 min (for a 6.5 kb plasmid)
I did the PCR reaction and ran the gel and got a band on the right size.
I then add 0.5ul DpnI and incubate at 37 degree for 1 hour. When I ran the gel after DpnI digestion, I saw no band.
Even I couldn' t see the band after DpnI digestion, I did PCR clean up using a kit.
After PCR clean up, I followed the protocol and did the transformation using DH5a competent cells.
My transformation protocol is:
- Aliquot 40µl of DH5a cells for each transformation into 1.5ml tubes that have been pre-chilled on ice
-Add PCR clean up product to DH5a cells and mix gently
-Incubate the tube on ice for 30 minutes
-Heat shock at 42°C for exactly 45 seconds
-Place tubes on ice for 1:30 minutes
-Add 250µl of pre-warmed (37°C) LB Broth
- Shake at 37°C for 1 hour
-Spread 200µl of each transformation onto LB plates with appropriate
antibiotic
-Allow plates to dry and incubate inverted at 37°C overnight
Result: I got no colonies!
I have been repeating this for the past 2 weeks and getting nothing.
The PCR seems to have worked well. I tried to transform my pre DpnI digested PCR product and got a lot of colonies.
I couldn' t get any colonies after the transformation of post DpnI digested PCR product.
Please, could anyone see where the problem is?
Thanks for any suggestions!
-Didem
The disppearing band suggests that the mutagenesis may not be working well enough. You are digesting away WT construct and there is no product left behind. A few thoughts on what might help:
How long are your primers? I seem to find that longer primers (40-45 bp) are better than shorter ones (25-30 bp).
Try more cycles. The Quikchange method is only a linear (as opposed to exponential) amplification of your vector. As I understand it, the main reason for using so few cycles is to minimise the potential for a mistake by the polymerase. This isn't a problem if you aren't getting amplification in the first place, so using more cycles shouldn't be harmful.
How much product are you adding to your competent cells? (I assume you are using chemically competent cells). Too much can be toxic, so reduce it down to 1 or 2 uL of PCR product.
Do you have other mtuagenesis primers that you know work? Perhaps running those at the same time could help shed some light on where the problem is.
Did you compare a DpnI digested and a non-digested product on an agarose gel?
The "lots of colonies" suggest that your template is quite enough to transform the cells, maybe you saw the template and the PCR didn't work at all.
true. the answers above make complete sense.
Ist totally possible. So I advise you to use megaprimers (upto 80-90 mer or atleast 45) to perform PCR. also, increasing cycle number, standardising PCR is the first step.
For the comp cells.. you can just in case use Commercial high Efficiency cells like NEB10.. TOP10
I had the same problem. But then I carried out a gradient pcr and found lower temperatures than previous reactions. and this helped.
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