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cloning problems using in-fusion kit


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#1 paularbbc

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Posted 08 April 2015 - 12:56 AM

I'm trying to clone as insert of 3kb in a vector of 7kb. I used the recombination kit of clontech (in-fusion), transform the bacteria but had no colonies. I've already tried everything suggested by the kit and also sequenced the product of PCR (insert) in the TOPO plasmid and everything seems to be fine. Can anyone help me?

 

 



#2 bob1

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Posted 08 April 2015 - 02:17 AM

Details will help - what have you actually done?



#3 miST32

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Posted 14 April 2015 - 05:52 PM

Hi paularbbc,

This technology has been pretty fool proof in my hands so far - sorry to hear it's giving you trouble.  Let's see if we can work through it.

First - some basics to check:
- Are you sure your cells are high-competency?

- Are you using the correct antibiotic?


Other questions:
- Did your positive control reaction (Linear pUC19 + insert) give you hundreds/thousands of colonies?

- How much DNA are you using per reaction? 

- What is the DNA source/purity and what is it suspended in?  Is the pH abnormally high or low?  How did you purify your insert?
I only ask because these reactions are so small, and contaminants or EDTA might be a factor if you are using relatively large volumes of DNA in the 10uL InFusion reaction.

- How did you design your homology regions in the primers for your insert?

- How much are you transforming, and what type of competent cells are you using?

- Are you using a restriction-linearized vector or a PCR-linearized vector?
--- If restriction-linearized, are they cohesive or blunt ends?
------ If cohesive, did you design the homology regions in your insert PCR primers to include the 5' or 3' terminal bases?  This matters for the resultant 3'-->5' exonuclease-derived complementarity between the insert and vector.

--- If PCR-linearized, are you purifying it?

Let us know more of the parameters in your setup so we can help you solve the problem.

Cheers

 



#4 Wek

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Posted 30 April 2015 - 08:04 PM

Sorry to hijack this thread but I rather not make another thread about Infusion.

 

I am trying to use Infusion for the first time to do some cloning. I made primers for my insert with the Infusion online tool, did the PCR and verified it by running it on a gel. Is it possible to do the infusion step with the PCR product as is? I gel purified my insert but I ended up recovering <300ng total and there's a lot of salt in it (low 260/230 but good 260/280).

 

I linearized my vector tonight and deactivate the RE. Can I skip the gel purification step and use whatever amount of vector I need after I nanodrop it again? I am sure Ill lose a lot of the vector and will and up with a shitty 260/230 ratio.

 

Btw, anyone knows approximately how much DNA you get from a 30-34 cycles reaction?It should be a few micrograms right?


Edited by Wek, 30 April 2015 - 09:38 PM.


#5 miST32

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Posted 01 May 2015 - 05:52 AM

Wek,

If your amplicon is clean (no nonspecific amplification), I would recommend column purification. Skip the gel purification to keep higher yields.

I usually do two 25uL reactions and column purify both in one column to achieve yields in the ug range.

I would not recommend using the PCR mixture itself. In theory the 3'-5' exonuclease and polymerase activity could fuse primers + amplicon + vector, but I suspect that this will saturate the enzymes with available reactive DNA "ends" and reduce your yield in practice.

Additionally, I would be careful introducing free nucleotides and salts from the PCR mixture to your tiny 10uL InFusion reaction.

*edited typo


Edited by miST32, 01 May 2015 - 06:55 AM.


#6 miST32

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Posted 01 May 2015 - 06:59 AM

Also - regarding the vector linearization step, if you are using a single restriction enzyme to linearize and are confident that you've achieved complete cleavage of your vector, then I think you could use it from a deactivated digest.  However, I might still consider purifying it on a column to get the DNA into water or tris instead of the digest mixture.






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