02 February 2016 5 9K Report

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

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