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Problem proving insert is in vector :(


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#1 Ms new scientist

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Posted 04 August 2009 - 10:44 AM

Hi everyone

I am having many problems with sequencing and proving my insert is in my vector!

My problem is (from the start):

I ligated and transformed my insert in a pmal p2x vector into SURE competent cells.

I get maybe 2 colonies per transformation plate which I then perform Spin Mini Prep from QIAGEN. Following their instructions, I grow the colony overnight in a LB-Carbenecillin 50ug/ml falcon tube. I then purify the plasmid using their kit and one strange thing I noticed was that when I measure the concentration of the plasmid extracted, it is low (50-80ng/ul) but then I send it for sequencing anyway and the sequencing results came back 'failed' with too many overlaping signals on the graph they gave me.

Also another thing I did was to perform colony PCR on some of the colonies and I get many bands (of higher and lower molecular mass than the insert). It is as though the bacterial genomic DNA underwent PCR.

I might try and reduce the number of cycles and increase annealing temperature for PCR but what else can I do? And also why did my sequencing results fail? It seems to be contamination (many overlaps) but from what?

Thank you so much for your help!

#2 phage434

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Posted 04 August 2009 - 02:16 PM

I'd guess your primer is bad. Is this a standard primer, or one you designed?

#3 Chakchel

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Posted 04 August 2009 - 11:24 PM

Hi,
I guess that your ligation or transformation is not working properly as you only get around two colonies per transformation. This is a quite low number. So maybe those colonies do not contain your insert - as the PCR results suggest, too. ;)

Did you also check your mini on a gel comparing it to the empty vector?

Greetings,
Chakchel

#4 pET

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Posted 05 August 2009 - 02:28 AM

I would do a control digestion of the prep.
pET

#5 perneseblue

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Posted 05 August 2009 - 03:51 AM

I agree with pET. Try doing a restriction digest first to confirm that your insert really is in your vector.

A successful transformation, typically will yield hundreds of colonies. As you are only getting two 2 colonies per transformation plate, I would be concern that the ligation has failed. Furthermore for a colony PCR, the presence of multiple bands in the PCR reaction, typically indicates that the transformation has failed. The region that you are amplifying is not present.

(Multiple bands in the colony PCR can also indicate that the primers are not working, but that is uncommon, although not unheard off)
May your PCR products be long, your protocols short and your boss on holiday

#6 HomeBrew

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Posted 05 August 2009 - 04:10 AM

I agree with the others -- I think your ligation is not working. Can you tell us how your prepared your insert and vector for ligation, and what your ligation and transformation protocols are?

I then purify the plasmid...[and] when I measure the concentration of the plasmid extracted, it is low (50-80ng/ul)...

Have you looked at the plasmid on a gel? Do a restriction digest of the extracted plasmids and compare them to a sample of vector only cut with the same enzyme(s) and a sample of uncut vector only.

...another thing I did was to perform colony PCR ...and I get many bands...

Try this PCR on the extracted plasmid. This will be much clearer than colony PCR.

I might try and reduce the number of cycles and increase annealing temperature for PCR but what else can I do? And also why did my sequencing results fail?

Tell us about your primers and your PCR conditions and thermal cycler program.




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