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pcDNA 3.1 Directional TOPO clone issues


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#1 Muzamil

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Posted 01 July 2014 - 09:34 PM

Hello, I have some problems with pcDNA 3.1 Directional TOPO cloning. I amplify 3 kb fragment from HIV RNA and try to insert it into the pcDNA 3.1 D/V5-His-TOPO vector. Transform the whole ligation mixture (6 microlitre) TOP10 competent cells. However, there are no or very few colonies on the plate.And most of the colonies are false positive. I need all of you experts' instructions. kindly help.

#2 bob1

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Posted 02 July 2014 - 01:08 AM

Details - how did you amplify, how did you confirm amplification?

How did you transform, in what volume, did you check the resistance markers?

What do your controls look like?

 

If you have a positive - i.e. it worked - what's the problem?  You only need one...



#3 Bio-Lad

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Posted 02 July 2014 - 01:45 PM

Hello, I have some problems with pcDNA 3.1 Directional TOPO cloning. I amplify 3 kb fragment from HIV RNA and try to insert it into the pcDNA 3.1 D/V5-His-TOPO vector. Transform the whole ligation mixture (6 microlitre) TOP10 competent cells. However, there are no or very few colonies on the plate.And most of the colonies are false positive. I need all of you experts' instructions. kindly help.

 

As Bob1 said, you only need one clone.  Assuming you didn't get any correct...

 

1) I'm curious why you are transforming the whole reaction.  Have you tried using only the 2uL recommended by the protocol?

2) What is the concentration of your PCR product.  Did you column purify it (or gel purify it if there were more than one product)?

3) How long did you let the reaction run?

4) Did you do a positive control for transformation (sometimes the cells just crap out over time).


Try pLOT, the a free plasmid mapping tool!

http://plasmidplotter.blogspot.com/


#4 Muzamil

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Posted 04 July 2014 - 08:04 PM

I amplified my gene of interest  using specific primers having CACC inthe forward primer Phusion Taq and checked the amplicon on agarose gel. Purified the product with column and got around 60  per microlitre concentration. Ligation was done using 10 nanogram of the insert and 1 microlitre of the vector (1:1) along with 1 microlitre salt solution. I used topo 10 comp cells and as per the protocol used the whole ligation mixture for transformation. I also took postive control and that worked. But most of the colonies are pseudo positive with my ligation .

kindly help.

 



#5 Bio-Lad

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Posted 05 July 2014 - 07:10 AM

I could be incorrect, but if you are using the Invitrogen directional cloning kit, the protocol says to use 2uL, not the whole reaction.  I'm not sure if that's part of the problem you are experiencing but if everything else you've done is exactly what the protocol stated, then I'd certainly to use 1-2uL and see if that improves your results.


Try pLOT, the a free plasmid mapping tool!

http://plasmidplotter.blogspot.com/





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