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Changing the vector

cloning

48 replies to this topic

#46 Bio-Lad

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Posted Yesterday, 03:20 AM

Again though, if you are already getting a visible band using your other conditions, simply doing a TOPO cloning may be the way to go.

Try pLOT, the a free plasmid mapping tool!

http://plasmidplotter.blogspot.com/


#47 RNA woman

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Posted Yesterday, 11:08 PM

For the Topo cloning. It is written as note to not use 5 phosphates primers for PCR that PCR product synthesised will not ligate. I don't understand why? I will remove the primers with the column and the PCR product it self will not have 5' phosphates. 



#48 phage434

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Posted Today, 03:22 AM

Your PCR product will have 5' phosphates if the primers have those phosphates. I was unaware of that issue with TA cloning, since I never use it. You can remove the 5' phosphates with shrimp alkaline phosphatase or antarctic phosphatase. Likely you can directly add 1-2 ul of SAP to your PCR reaction following PCR, without purification. AAP will require adding its buffer, which contains zinc.



#49 Bio-Lad

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Posted Today, 03:32 AM

I never knew that either. I was just looking it up. You learn something new every day!

Try pLOT, the a free plasmid mapping tool!

http://plasmidplotter.blogspot.com/






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