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PCR screening of transformed bacterial colony


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#1 xyz

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Posted 15 June 2009 - 02:08 PM

Hello everyone,
I had transformed the desired clone and got nice separated colony. Before sequencing them I want to screen the colonies. For that I chose the forward primer of my gene and reverse primer lies in the middle region of gene(Is this a good strategy?). Is 20microloiter PCR reaction mix is fine to screen the colonies? After PCr amplification how much volume should I load in the thin combed agarose gel? I tried with 10microliter of DNA ladder in the gel, but very faint resolution. Any suggestions--------------

#2 Rsm

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Posted 15 June 2009 - 04:23 PM

Which primer you use depends on what you want to do with the plasmid. If you want to use it for normal cloning of your gene, you can use internal primer like yours. If you want to overexpress your gene, and you need the correct orientation, you might want to use a forward primer on the plasmid and a reverse primer on your gene. Positive colonies will have the right orientation.
20ul reaction mix is sufficient to screen colonies. You need to pick the colonies and put them into the H2O needed for your PCR reaction (15ul?), and then into LB medium until your PCR finishes. Then you can conveniently expand your positive colonies. I strongly recommend to use a positive control, e.g 5ng of a vector containing your gene.
I usually use 4ul of PCR product (30cycles) for the gel. If you have used 10ul DNA ladder and got faint staining, it seems your gel does not contain EtBr...
Cheers,
Minna
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#3 jiajia1987

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Posted 15 June 2009 - 10:33 PM

I usually screen by doing a colony PCR with one primer that starts from the 5' end of my insert and a reverse primer that is from my plasmid. The volume would be 30ul. Usually load like 3 to 5ul of the PCR products on the gel. and, if you want an easier time, you can use GelStar, which is pretty sensitive and allows the band you want to shine up very easily under blue transilluminator light or UV light.

#4 xyz

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Posted 16 June 2009 - 09:59 AM

Thanks a lot.

Which primer you use depends on what you want to do with the plasmid. If you want to use it for normal cloning of your gene, you can use internal primer like yours. If you want to overexpress your gene, and you need the correct orientation, you might want to use a forward primer on the plasmid and a reverse primer on your gene. Positive colonies will have the right orientation.
20ul reaction mix is sufficient to screen colonies. You need to pick the colonies and put them into the H2O needed for your PCR reaction (15ul?), and then into LB medium until your PCR finishes. Then you can conveniently expand your positive colonies. I strongly recommend to use a positive control, e.g 5ng of a vector containing your gene.
I usually use 4ul of PCR product (30cycles) for the gel. If you have used 10ul DNA ladder and got faint staining, it seems your gel does not contain EtBr...
Cheers,
Minna



#5 xyz

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Posted 16 June 2009 - 10:00 AM

Thanks for reply

I usually screen by doing a colony PCR with one primer that starts from the 5' end of my insert and a reverse primer that is from my plasmid. The volume would be 30ul. Usually load like 3 to 5ul of the PCR products on the gel. and, if you want an easier time, you can use GelStar, which is pretty sensitive and allows the band you want to shine up very easily under blue transilluminator light or UV light.



#6 liweixie

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Posted 21 June 2009 - 09:44 AM

Hello everyone,
I had transformed the desired clone and got nice separated colony. Before sequencing them I want to screen the colonies. For that I chose the forward primer of my gene and reverse primer lies in the middle region of gene(Is this a good strategy?). Is 20microloiter PCR reaction mix is fine to screen the colonies? After PCr amplification how much volume should I load in the thin combed agarose gel? I tried with 10microliter of DNA ladder in the gel, but very faint resolution. Any suggestions--------------

25 ul is enough.
It is just a screening and you can load all of the screening reaction on the gel.

#7 Qundo12

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Posted 22 June 2009 - 03:03 AM

Hello everyone,
I had transformed the desired clone and got nice separated colony. Before sequencing them I want to screen the colonies. For that I chose the forward primer of my gene and reverse primer lies in the middle region of gene(Is this a good strategy?). Is 20microloiter PCR reaction mix is fine to screen the colonies? After PCr amplification how much volume should I load in the thin combed agarose gel? I tried with 10microliter of DNA ladder in the gel, but very faint resolution. Any suggestions--------------

25 ul is enough.
It is just a screening and you can load all of the screening reaction on the gel.

20 ul is enough (i usually use that), but I don't think 10ul of ladder is not enough because normally companies provide the ladder with quite high concentrations and about 5ul is enough.
Be careful because you can get the "false positive" colonies. PCR is very sensitive, it can amplify the remaining free inserts on the plate (to recognize the fake one, the band observed is lower in brightness compared with positive control).
To avoid this, I strongly recommend you to transfer colonies to master plate (especially if you use Ampicilline as the selective antibiotics), because you can almost exclude the remaining particles of ligation reaction on the plate.




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