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Failure to BP Clone. Troubleshooting advice? Switch to TOPO TA cloning?


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#1 Hotspot17

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Posted 24 October 2013 - 11:35 AM

Hi everybody,

My first post here on this forum. Been BP cloning all of last year a number of different PCR products with the gateway system with no problems but suddenly everything stopped working. I already got new enzyme, use newly extracted plasmid pDONR201, and check my PCR products for the correct sequence. In the past I normally didn't try the positive control but multiple tries resulted in failure to get bacterial colonies. Are there any ways to troubleshoot this problem? 

 

My current strategy was to BP Clonase a PCR product into an entry vector (pDONR201) then LR clonase the entry vector into pDEST15 for bacterial expression. Would switching to TOPO TA cloning be a viable option to replace the BP clonase? Any help would be greatly be appreciated. Been banging my head at his for 8 months now with no luck. 

 

Addition information:

Entry vector plasmid: pDON201

Destination vector plasmid: pDEST15 for bacterial, and pEGHA for yeast

PCR products contain the gateway sequence designed into the corresponding primers.

Use 1 ul of the BP clonase reaction to transform 50 ul of the competent cells.

Heat shock for 30 secs --> 42 C then 2 mins on ice --> Add 400 ul of SOC media and shake at 37 for an hour --> lightly spin down tube to concentrate cells and plate onto Kanamycin plate --> 37 C overnight



#2 phage434

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Posted 24 October 2013 - 12:00 PM

It sounds as if your competent cells are no longer competent. Time to measure their competence by transforming very small amounts of known good plasmid. You should have efficiences of at least 10**8 cfu/ug of plasmid. So, if you transform 10 pg of plasmid, you should get at least 1000 colonies.



#3 Lilito

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Posted 29 October 2013 - 12:18 PM

Hi,

 

I had the same problem, the only difference is that I used pDonr221. I was stuck for months especially at the LR step. I never really found out what was the reason but what I did was just transform bigger volume of cells. I got several colonies for the BP step and 2-3 colonies for the LR. I think for some reason DH5alfa didn`t really like these plasmids. The transformation I did was as follows:

add 1 microl BP or LR reaction in 150 microl bacteria;

on ice for 5 min, 

heat shock 45 secs,

back on ice for 30 min,

850 microl Lbroth,

shake at 37 for 1 hour,

spin down and plate 

 

Good luck!!!smile.png






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