subcloning of PCR product, please help! - subcloning problems (Jun/07/2005 )

What is supposed to be a piece of cake, cohesive end subcloning has not worked for me in two months, and I am getting desperate!
I generated EcoRI and BamHI restriction sites on my insert (800 bp), the restriction sites are 3-4 bp from the ends.
I am sucloning into a pGBKT7 (Clontech) vector (7.3 kb), cut with the same enzymes.
I tried virtually all posible conditions, modifications, troubleshooting, controls for ALL steps involved (i.e., PCR amplification, restriction digestion, ligation, transformation).
Positive controls work at each stage. The colonies that I get occasionaly NEVER have an insert in them.
Any suggestions will be greatly appreciated!

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Hi innabobolina,

I have had a similar problem in the past. From it I learnt, no matter what the company literature says restriction enzymes do not cut close to ends 3 or 4 bases is rarely enough.

What I'd suggest is blunt end cloning it into another cloning vector and then cutting from there and cloning it into the pGBKT7 vector. When I tried that it worked first go.

Hope this helps,

Scott

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hi
according to neb
EcoR I cutting of these fragments is >90% (1bpadded to restriction site seems enough) after 2 h...
----GGAATTCC
--CGGAATTCCG
CCGGAATTCCGG

For BamHI :
CGGATCCG 10% after 2h
CGGGATCCCG >90% after 2h
CGCGGATCCGCG >90% after 2h

hence i would not say it's a matter of close-to-the-end-cutting.
Try to greatly increase the ratio insert vecteur (1:15 may be necessary).

do you make a negative control? (ligation whithout insert) ?

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Hey all,
I've got a similar problem with cloning. I want to clone a 3.4 blunt end PCR product into a 2.6kb pTRE tight vector. First I tried blunt end cloning and got colonies...but they never had the insert. Then I used for the PCR of the 3.4 kb fragment Primers with a NotI and SalI restriction site (each of the Primers have additionally 6 bp in distance to the restriction site) and tried it with cohesive end ligation. Thus I digested the vector with the appropriate restriction enzymes, dephosphorylated the vector and did the ligation in different molar ratios (up to 1:8 insert: vector). Before transformation I also run a gel of the ligation mix (also the vector/vector and insert/insert controls) and could see the successfully ligated 6kb band on the gel. I used XL2 blue cells for transformation and again got quite a lot of colonies. But again....screening these colonies with appropriate restriction digests I never got the bands I would expect in a successfully transformed cell. There seems to be something wrong with the cells....maybe rearrangement within the cell?
I'm also quite desperate, so please help us with this problem, thanks!
,- prize laureate

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I've just gotten over similar cloning problems. I was PCR'ing with EcoRI/XbaI sites at the end. Went from 5bp to 10bp end piece and still couldn't digest and ligate into my target vector.

I ended up taking the PCR product into a TOPO vector then cutting out of there and ligating it into my original destination vector. Worked like a charm.

Technically digesting the PCR product should be no problem, and I have done it many times. But once and awhile all goes to bunk.

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need to check the efficiency of the double disgestion:
For vector, after doube digestion, do a self ligation and transformation, positive results will be very few colonies.

For insert, do self ligation and run an agrose gel, positive result will be a ladder (monoer, dimer, trimer ....). if you only see monermer, dimer, that means one of the enzyme didn't cut.

Lastly, what are you trying to clone? is is poisonous to cell ( endonucleases for example??)

good luck

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I am having the same problem as innabobolina. When you do blunt end cloning using a PCR product, do you dephosphorylate the vector? I am not sure if PCR product ends are phosphorylated or not.

Thanks!

Roshni

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I second what Scott said. First do a TA cloning, then cut out the insert and do a subcloning.

Even the vector has incompatible ends after double digestion, I remember somebody said here that it is better to dephosphorylate it otherwise you will spend the rest of your life screening colonies.

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[quote=Roshni29,Jun 7 2005, 02:51 PM]
[/quote]


I am having the same problem as innabobolina. When you do blunt end cloning using a PCR product, do you dephosphorylate the vector? I am not sure if PCR product ends are phosphorylated or not.

Thanks!

Roshni

[/quote]

Hi Roshni,

PCR product is not phosphorylated and therefore in blunt end cloning must be treated with T4 polynucleotide kinase. Also when doing blunt end cloning I ALWAYS dephosphorylate vector, I also do this even with non-compatible cohesive end ligations.

Finally as pcrman said the easiest way to do the initial cloning is do a TA cloning, but that depends on the polymerase you use being able to generate a terminal A overhang (Taq does it). I commonly use a mixture of Taq and proofreading enzyme this has the advantage of lower misincorporation rate and still enables TA cloning, most companies have a version of this.

Hope this helps.

Scott

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Hi Innabob,
I was happy to see your post because I have been trying to subclone a 1.2 kb insert into pGBKT7 for a little over a month using BamHI and NdeI cohesive ends. Just like you, I've got Bam and Nde sites in my primers when I amplify up my insert, but when I digest, ligate and transform I get no inserts from any colonies!
Just last week I tried blunt-end cloning into the pCR 2.1 commercial vector, which is specifically designed for cloning PCR products. It comes linearized with T overhangs; since PCR products naturally have A overhangs it should ligate right in. However, I only got 3 colonies and none of them had the insert. At this point (if my PI isn't fed up with my working on this project), I suppose I will try it again with the kinase/phosphatase treatment of the insert and vector as people were suggesting in the forum.

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