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Site-directed Mutagenesis


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#1 erichaines13

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Posted 22 October 2010 - 11:36 AM

Hi, I have been trying to do two single point mutations in the EGFR in pcDNA3.1 vector (~9.5Kb) without any success. If I run my product on a gel I can see a product before and after DpnI digestion suggesting that my PCR is working. However, everytime I try to transform, either in XL Blue or DHalpha cells I get no colonies. A colleague suggested that my bacteria might now be competent enough to allow for the recombination of my DNA to occur. What do you think?

PCR reaction:
100ng DNA template
500ng Primer
1ul DNTP mix
5ul PFU turbo buffer
1ul PFU turbo
Fill to 50ul with water

PCR program

1- 95C 5 minutes
2- 95C 45 seconds
3- 52C 45 seconds
4- 72C 18 minutes (repeat 2-4 20 cycles)
5- 72C 10 minutes

I digested 2 hours at 37C using 2,5units of DpnI

Please help me

Thanks
Eric

#2 pDNA

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Posted 22 October 2010 - 12:17 PM

check the competency of your cells ...if you prepare them yourselfe ...or go and buy some with a defined competency.

You will need a really good competency (i would assume 10E8 or 10E9) due to the fact that the product DNA must undergo the nick repair process in E. coli and is not supercoiled, resulting in reduced efficiency of bacterial transformation.

Regards,
p

#3 GNANA

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Posted 22 October 2010 - 12:22 PM

Did you purify ur DPn digested product before transformation?....if not do gel purification and transform,,, doing this u fail, for sure the prob is with ur competent cells.

good luck.
Gnana...
I would prefer being perfectionist rather than a passionist in Research.

I always had an alternate hypothesis....

#4 donny

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Posted 24 October 2010 - 01:35 AM

I noticed you're using more template and primers than recommended for your reactions. Any reasons for this? Try using less primers and template. I used only 0.2uM and they seem to work much better than 1uM. I guess it may have something to do with depleting the dNTP supply too fast, especially when amplifying such a large construct. Using more template will lead to more amplicon-template hybrids which will be digested by DpnI as well, thus reducing your yield. There seems to be different recommendations for the extension time and temperature of Pfu Turbo. For usual PCR, they recommend 2min/kb for target >10kb at 72C but 1min/kb at 68C for Quikchange (another version of Quikchange manual says 2min/kb!). I dunno which to trust. Maybe you can try them out. Excessive extension time may be deleterious to PCR products. You may also want to reduce your denaturation time to reduce DNA damage.

#5 erichaines13

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Posted 25 October 2010 - 12:07 PM

Thanks for your advice. I decided to test other lab's competent cells to see if I have anymore luck with them. At the same time I am making fresh competent cells. I have used the same protocol in another laboratory and and worked perfectly there! So I really think its the lab's competent cells. I am going to make fresh ones the same way I made them in my other lab. Just another quick question: I tried the control reaction suggested my stratagene (control primers and template) and my product is like 20 times more abundant in the control reaction compared to the experimental reactions. Is that normal?

#6 hematopoietry

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Posted 26 October 2010 - 02:07 AM

If you have low competent cells and time is more of an issue than money, do your PCR with 10 times as much, transform to 10 times as much bacteria as usual (and grow in larger volume), spin down and remove most of the sup, resuspend and plate everything.

Edited by hematopoietry, 26 October 2010 - 02:09 AM.


#7 erichaines13

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Posted 28 October 2010 - 07:25 AM

I made a fresh stock of competent cells and transformed. To my pleasure I obtained multiple colonies!!!! Hopefully they are positive! Thanks Everyone

#8 erichaines13

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Posted 04 November 2010 - 03:58 AM

Got another problem...So I mini-prepped the colonies I obtained from the new competent cells I made and sequenced 4 of them. None of the colonies were positive for my mutation. All my sequences came back as wild-type except for one in which the sequencing was unable to read the residue that was suppose to be mutated. Why did my mutagenesis not work? Should I test the construct in which the residue was unable to be sequenced by western just in case it is positive? Is it worth sequencing the other clones that I isolated?




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