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cloning problem


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#1 genugene

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Posted 25 October 2004 - 10:24 PM

I am trying to clone a gene(2kb) in pEGFP vetor. for that I used Hind III and Pst I in primers and added bases recomanded by NEB to their 5'- ends. I got very good PCR product and purified it with QIAGEN kit (checked after purification). Digested insert and vector and again purified for ligation. But I didnt get a colony, when the ligated products were transformed in BL21 and XL-1 blue cells.
Then I tried T-A cloning(MBI) and got few colonies. But surprisingly during isolating plasmid, while i am trying to spin down the cells(XL1-Blue), it is appearing very sticky/slimy and this pellet is impossible to resuspend with GTE(Sol-I). I am not getting any plamid through either phenol-chloroform or PEG method.
Can anyone help me?
Thanks

#2 biomed

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Posted 26 October 2004 - 08:13 AM

I am trying to clone a gene(2kb) in pEGFP vetor. for that I used Hind III and Pst I in primers and added bases recomanded by NEB to their 5'- ends. I got very good PCR product and purified it with QIAGEN kit (checked after purification). Digested insert and vector and again purified for ligation. But I didnt get a colony, when the ligated products were transformed in BL21 and XL-1 blue cells.
Then I tried T-A cloning(MBI) and got few colonies. But surprisingly during isolating plasmid, while i am trying to spin down the cells(XL1-Blue), it is appearing very sticky/slimy and this pellet is impossible to resuspend with GTE(Sol-I). I am not getting any plamid through either phenol-chloroform or PEG method.
Can anyone help me?
Thanks

Hi,
1. First of all, did you get successful ligation? No clear.
2. what transfection method did you use? I used FuGene to transfect pEGFP and get a good result.
3. check your lysis buffer for oyur prep. Sound like wrong buffer.

#3 loustau

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Posted 27 October 2004 - 04:56 AM

Hi,

It might sound stupid, but are you sure about which selection to use? i.e. ampicillin/kanamycin. Even in the best of cases, I have few colonies in the negative control, so when I don't see a single one, it's usually because I used the wrong selection. That has also caused those hard to resuspend bacteria and no DNA yield. For some reason, these pellets also seem whiter than usual, no?

#4 dmarial

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Posted 01 November 2004 - 09:24 PM

Hi,
I think that the problem you are having is resulting from the strains of bacteria you are using. For cloning purposes DH5alpha cells are the most common type of bacteria used. BL21 and XL-1 cells are used for recombinant protein synthesis and purification and as such contain mutations (as well as lysing enzymes) that enhance the production and purification step. Therefore, the cells lyse very easily which is causing the very sticky/slimy goo that you are seeing. Try using DH5alpha cells ;)

#5 genugene

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Posted 01 November 2004 - 10:52 PM

Hi
dmarial
thanks for your response. but the supplier of the pEGFP has recomanded BL21 strain for the purpose(DH5alpha is also there in there list but we dont have). ;)




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