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cloning help; high salt level in vector


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#1 luckyjade

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Posted 13 September 2013 - 09:00 AM

Hello,

 

I just checked my plates this morning and absolutely zero colonies grew. I was wondering if anyone has any ideas as to what could be the problem. Below are some things that may have gone wrong. I checked the digested vector and insert and they are in fact the right size. I'm led to believe that the problem is with ligation/transformation

 

-I used a Qiagen gel extraction kit to get my vector. When I measured the od, there was a high peak at 230 for salt levels. Am I able to just get rid of this with a PCR purification kit?

-I did a 20uL ligation reaction. 15 minutes at room temp.  I used 5 uL of that with 50 uL competent cells. 15 minutes in ice. 30 seconds in 42 degree incubator. 2 minutes back in ice. Added 50 uL LB. Warmed up plates for about 30 minutes prior to plating using glass beads.

 

The competent cells that I have used have been tested and have high efficiency. Vector size is 6000 and insert size is 1000 and I used 3 fold molar excess for ligation.

 

Any help is much appreciated, thanks in advance.



#2 phage434

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Posted 13 September 2013 - 11:36 AM

The added 50 ul of LB is (1) far too little and (2) not the optimal medium.  You should be adding at least 500 ul of SOC buffer, and then growing the cells with shaking at 37 for an hour. The easiest way to do this is to do the transformation in a 2 ml tube, which allows good aeration during shaking (horizontally).  I've never found warming the plates to make much difference, but it couldn't hurt.

 

Many other things could also be wrong. Have you measured the competence of your cells? The 230 nm peak usually indicates Gu-HCl contamination, not salt. This could be a problem, and yes, it can be removed with a spin column. Easier, perhaps is just to precipitate, wash, and resuspend your DNA.

 

I would use 2 ul of ligation in your transformation -- large amounts are inhibitory.



#3 scolix

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Posted 14 September 2013 - 11:47 AM

I agree with phage434. You have to shake the culture after you add SOC media. 



#4 luckyjade

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Posted 18 September 2013 - 09:41 PM

Thanks for your responses! I forgot to add that the vector is amp-resistant so no 37 degree incubation/shaking is necessary. what else would you guys suggest doing? i've tried just 2 uL of ligation reaction and still no colonies

 

i am using competent cells from the same batch that i made about a month ago. all my previous cloning has worked with high efficiency and i have tested the competent cells with just plasmid. i have a feeling that the problem may be my with my ligase or ligase buffer. is there any way to check if my ligation reaction was successful? is it possible to run a bit of the ligation reaction on gel through gel electrophoresis? what would a control for that be? just the digested vector and see if my ligation reaction has the added insert's size?


Edited by luckyjade, 18 September 2013 - 10:18 PM.


#5 phage434

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Posted 19 September 2013 - 04:35 AM

I routinely do the outgrowth with amp, even if not strictly necessary. The efficiency is higher, and I don't have to think about what antibiotic I'm using (I often mix antibiotic resistances).

But the low added medium is a serious problem (and the choice of SOC would be better).






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