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Mutagenesis

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#1 MaryNelson

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Posted 13 May 2015 - 01:13 PM

Hi all.  I've been trying to use the Agilent QuikChange XL Site-Directed Mutagenesis Kit, and haven't been getting any PCR product on a gel after performing DPN digestion.

 

My primers were designed using http://www.genomics....signProgram.jsp

 

GC content ranges from 48% to 54%, melting temperature is greater than 78C using the agilent equation, and length ranges from 46-54 bases.  Primers are complimentary, as per the Agilent protocol.

 

I'm trying to make five different mutants, each separately, each with 5 bases substituted, and none of the 5 primer pairs has worked.  Since it's not just one pair of primers failing, and they were all designed using the recommended program, I'm skeptical that primer design is the problem.

 

My plasmid is about 5kb in size.

 

My standard reaction uses

5ul 10x reaction buffer

125 ng of each primer

1ul of dNTP mix

3ul of quicksolution

1ul of pfuTurbo polymerase

ddH2O to bring to 50ul

for template I've tried 5, 10, 20, 50, and 100 ng (while maintaining 125 ng of each primer)

I've also tried adding 1.5uL of DMSO

 

 

I've used the recommended cycling parameters:

* 1 cycle of 95C for one minnute

* 18 cycles of 95C for 50 seconds, 60C for 50 seconds, 5.2 minutes of 68C

* 1 cycle of 7 minutes of 68C

 

I've tried using fresh dNTPs and new pfuTurbo polymerase.  Switching to a different kit isn't really an option (we have several of the Quikchange kits).

 

Any and all assistance is appreciated.


Edited by MaryNelson, 13 May 2015 - 01:45 PM.


#2 Micro

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Posted 13 May 2015 - 02:59 PM

Hi,

 

Just to clarify does each of you primers contain 5 base substitutions or are you introducing a single base substitution with 5 primers? 

 

Could you include your transformation methods? I've recently been using this kit and had the process fail due to errors in transformation process. Did you do both of the suggested controls? What were the results of the controls?

 

Cheers

Micro



#3 phage434

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Posted 13 May 2015 - 05:25 PM

You may not see a gel band. You should go ahead with transformation, even though you can't see a band on a gel after your PCR reaction. With only 18 cycles, limited amplification occurs in the pcr reaction. You should also verify that you have sufficient template for your reaction. Unlike a normal pcr reaction, this reaction requires relatively large amounts of template.



#4 MaryNelson

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Posted 13 May 2015 - 06:50 PM

Hi,

 

Just to clarify does each of you primers contain 5 base substitutions or are you introducing a single base substitution with 5 primers? 

 

Could you include your transformation methods? I've recently been using this kit and had the process fail due to errors in transformation process. Did you do both of the suggested controls? What were the results of the controls?

 

Cheers

Micro

 

I have 5 primer pairs, and each pair substitutes five bases (I'm testing the different effects of 5 different mutations... or I would be, if I could get this to work).

 

For transformation, I thaw ~200uL of chemically competent cells on ice, then add 200uL of 0.1 M CaCl2, then add the ligation mixture (after 2hrs DPN digest).

 

I incubate on ice for 30 minutes, heat shock at 42C for 2 minutes, then put on ice for exactly 10 minutes, followed by transfering the entire mix to a tube with 3mL LB broth which I shake at 37C for 45 minutes.  I then plate 100uL of cells on LB and also centrifuge 1mL of cells at 13,000rpm, resuspending in 100uL, and plate that too. 

 

The protocol and cells have always worked quite well for me in the past.

 

You may not see a gel band. You should go ahead with transformation, even though you can't see a band on a gel after your PCR reaction. With only 18 cycles, limited amplification occurs in the pcr reaction. You should also verify that you have sufficient template for your reaction. Unlike a normal pcr reaction, this reaction requires relatively large amounts of template.

 

I should have stated that I do go ahead and attempt transformation after my PCR reaction.

 

How much template would you recommend?  I've tried 10ng, 20ng, 50ng, and 100ng.



#5 labtastic

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Posted 14 May 2015 - 09:59 AM

Some thoughts....

 

In terms of getting better transformation efficiency:

 

I would suggest using the competent cells at full strength. Plus, cells made with just CaCl2 are not very competent. Consider other cell preparations, or use commercially prepared cells if they are available.

 

Also consider using SOC media to recover your cells instead of LB.

 

PCR cleanup your reaction after DpnI digest and elute in water. This will get rid of everything in the PCR reaction that interferes with the transformation (salts, detergent, protein, etc)

 

 

In terms of getting your PCR to work:

 

Try lowering your annealing temp to 50-55C, or use a gradient from 45 to 65C and see which works best

 

Use a shorter annealing time (30sec) and/or shorter denaturation time (30sec)

 

Order different primers. The ones that are spit out by agilents website seem to be primarily optimized for 80C annealing temp. This does not always produce the ideal primers. Try making them manually. A decent rule of thumb is add ~20 bases upstream and ~20 bases downstream of your mutation(s). 

 

If all else fails:

 

Do the mutagenesis by overlap extension PCR. Amplify using touchdown PCR in first round. Use slowdown PCR for second round to fuse the two halves and use nested primers if possible. This method is a bit more labor intensive, but pretty much always works.

 

When it comes to mutagenesis, my strategy is: Try quickchange one time. If it works, great. If not, don't troubleshoot the quickchange, go right to overlap extension because by the time you've done all that troubleshooting for the quikchange reaction, which may or may not help, you could have finished the overlap extension and moved on with the experiment.


Edited by labtastic, 14 May 2015 - 10:03 AM.






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