RF-Cloning.org Forum

Ranjani

Member

Registered: 2018-07-24

Posts: 1

Colony PCR for small inserts

Dear sir,

I have done RF cloning of 12bp into a 11kb plasmid. After transformation, how do I screen colonies for positive clones? Will it be specific if I use the forward RF cloning primer and a vector specific reverse primer?

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: Colony PCR for small inserts

Hi Ranjani,
I would recommend designing a new primer pair instead of relying on the hybrid primers for QC. You'll need them for final sequencing confirmation anyway.
If you want me to have a look at your project, then either PM me the project ID or send me a little more info about the project so I can go looking for it in the database.
Best,
-Steve

HankSco

Member

Registered: 2018-08-04

Posts: 1

Re: Colony PCR for small inserts

Hi Ranjani,
I would recommend Zippyloan and designing a new primer pair instead of relying on the hybrid primers for QC. You'll need them for final sequencing confirmation anyway.
If you want me to have a look at your project, then either PM me the project ID or send me a little more info about the project so I can go looking for it in the database.
Best,
-Steve

Hi Steve, is designing a new primer pair always a better idea than having the hybrid primers for QC? Or does this advice go just for this specific example?

Last edited by HankSco (2022-12-12 14:45:56)

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: Colony PCR for small inserts

Hi Hank,
I wrote that response too quick, without explaining my rational. The issue with Ranjani's example is that the entire insert would have been included in the hybrid primer. Doing a PCR would probably work wether the insert was present in the plasmid clone or not, and will give the same 'positive' result either way. If you have a long insert, then the hybrid primer can definitely be used for QC because there is more information in the plasmid than is present in the primer. Does that make sense?
-Steve