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PCR-based Site-directed mutagenesis


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#1 saltycookie

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Posted 27 February 2009 - 05:06 AM

Still I have problems on site-directed mutagenesis. I used stratagene QuikChange system. According to the manual, I examined the site-directed mutagenetic products on the gel. There were clear bends showed on the gel. Based on my previous experiences, once you see the bends on the gel, you have over 90% opportunities to get the clones. However, I got no colony after transformation. My competent cells were fine and the transformation procedure was just same as before. Do you guys have any idea about my situation? By the way, something on the troubleshooting part of the manual I cannot understand "Nicked or linearized plasmid DNA will not generate complete circular product. Verify that the template DNA is at least 80% supercoiled." Can somebody kindly explain me? How to verify that the template DNA is at least 80% supercoiled? Should I run another gel after DpnI treatment? Really confused.

Thank you.

#2 T C

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Posted 27 February 2009 - 05:10 AM

Hey

I have found this helpful

1. Decrease the amount of DNA you take for transformation, somehow salts inhibit transformation.
2. Column purify and get the DNA in water and then electroporate.

Best
TC

#3 saltycookie

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Posted 27 February 2009 - 05:48 AM

For the amount of DNA, I tried both high and low. Usaully I would use the DNA for transformation with less than 1/10 volume of competent cell. I think it should be fine. However, I got no colony in both cases. Anyway, I will try to perform column purification after DpnI digestion and then make a transformation. Thanks a looooot!

Cookie

[quote name='T C' date='Feb 27 2009, 09:10 PM' post='17137']
Hey

I have found this helpful

1. Decrease the amount of DNA you take for transformation, somehow salts inhibit transformation.
2. Column purify and get the DNA in water and then electroporate.

Best
TC

#4 NemomeN

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Posted 27 February 2009 - 03:27 PM

"Nicked or linearized plasmid DNA.." is referring to the template you are using for your PCR. Supercoiled DNA runs faster then relaxed DNA....run a linear sample next to your prep and see how much is supercoiled.

Also, more than 100ng of total DNA will affect your transformation efficiency. Since the resulting DNA from the extension is linearized, you will have a low transformation efficiency to begin with. Therefore, it is really important to make sure that you don't start with too much template or your transformation will suffer (Dpn1 digested DNA + amplified DNA should be less than 100ng, and that PCR buffer also reduced transformation efficiency). Lastly, are you using commercial or homemade competent cells? You need something with at least 10^6 but preferably 10^7 colonies/ug.

Good luck.

#5 T C

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Posted 27 February 2009 - 08:40 PM

Hey

Electroporation has higher efficiency than transformation so electroporate. You need to have DNA in water for electroporation.

Best
TC

[quote name='saltycookie' date='Feb 27 2009, 07:18 PM' post='17141']
For the amount of DNA, I tried both high and low. Usaully I would use the DNA for transformation with less than 1/10 volume of competent cell. I think it should be fine. However, I got no colony in both cases. Anyway, I will try to perform column purification after DpnI digestion and then make a transformation. Thanks a looooot!

Cookie

#6 scolix

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Posted 28 February 2009 - 08:07 AM

Some vectors sequences are GC rich and this can cause problems. Try to add DMSO in your reaction mix and give it a shot.

#7 saltycookie

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Posted 02 March 2009 - 01:38 AM

I indeed added DMSO in PCR reaction. Then I examined the mutagenetic PCR products on the gel and I saw an clear bend afterwards.

Some vectors sequences are GC rich and this can cause problems. Try to add DMSO in your reaction mix and give it a shot.



#8 saltycookie

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Posted 02 March 2009 - 01:54 AM

The mutagenesis which I performed was on a vector. I am sure that the vector was in a supercpiled form.
I think I can avoid this problem.

Thanks for those useful suggestions. I will take care of the DNA amount and quality of competent cells for transformation.


Cookie

"Nicked or linearized plasmid DNA.." is referring to the template you are using for your PCR. Supercoiled DNA runs faster then relaxed DNA....run a linear sample next to your prep and see how much is supercoiled.

Also, more than 100ng of total DNA will affect your transformation efficiency. Since the resulting DNA from the extension is linearized, you will have a low transformation efficiency to begin with. Therefore, it is really important to make sure that you don't start with too much template or your transformation will suffer (Dpn1 digested DNA + amplified DNA should be less than 100ng, and that PCR buffer also reduced transformation efficiency). Lastly, are you using commercial or homemade competent cells? You need something with at least 10^6 but preferably 10^7 colonies/ug.

Good luck.






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