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Problem with Cloning- NO colonies at all - (Nov/23/2007 )

I am trying to clone a 1.6 kb fragment into pcDNA3 expression vector (5.4 kb). I am cutting with 2 restriction enzymes, BamHI and XbaI.
When I run the gel of the restriction digests the position of the bands show that both enzymes are cutting the vector. I cut out the bands then use a QIAGEN gel extraction kit to extract the DNA.

My ligation reactions are:
1. pcDNA3 double digest only (negative control)
2. pcDNA3 double digest with insert double digest
3. pcDNA3 BamHI single digest only (positive control)
4. pcDNA3 XbaI single digest only (positive control)

When I transform the above reactions usings JM109 cells I get no colonines on any of the plates, not even the single digest vectors. The only way I get colonies is if I only use uncut vector in the transformation. Also, I have tried blunt end cloning into the transfer vector pT7blue and that works fine. I get lots of white colonies and colony PCR has shown that they contain the insert. I don't think the problem is vector specific because I have tried to put the same insert into pEGFP-N1 and have exactly the same problem (ie no colonines even on the positive controls). Also the ligase definitely works because it is the same one I use for pT7blue cloning.

Does anyone have any suggestions on what could be going wrong? Could something be digesting away the cut ends of the vector so they cannot religate?

-ksmith-

Common problems: UV exposure of the DNA while imagining and cutting bands. Use long (365 nm) wave UV or blue light and limit exposure.

Poor transformation efficiency: Measure transformation efficiency of your cells and protocol. It should be at least 10**8 transformants/microgram of plasmid DNA.

Ligation concentrations: Using too high a concentration of vector and insert leads to concatamers rather than closed circular DNA. Concentrations should be < 20 ng/ul.

Ligation amounts: Use 1-3 ul of ligation to transform 50 ul of competent cells. More is inhibitory.

Outgrowth of cells: For tet and chloramphenicol resistance, use 1.5 - 2 hour outgrowth in nonselective medium rather than 1 hour to improve transformation efficiency. 1 hour works well for Kan and amp.

-phage434-

Have you created a dam methylation site in your insert at the Xba I site? That doesn't explain the ligation 3 result but it explains ligation 2 and 4 results and the failure in the other vector. Nice post btw.

-killerkoz17-

QUOTE (ksmith @ Nov 23 2007, 03:44 AM)
I am trying to clone a 1.6 kb fragment into pcDNA3 expression vector (5.4 kb). I am cutting with 2 restriction enzymes, BamHI and XbaI.
When I run the gel of the restriction digests the position of the bands show that both enzymes are cutting the vector. I cut out the bands then use a QIAGEN gel extraction kit to extract the DNA.

My ligation reactions are:
1. pcDNA3 double digest only (negative control)
2. pcDNA3 double digest with insert double digest
3. pcDNA3 BamHI single digest only (positive control)
4. pcDNA3 XbaI single digest only (positive control)

When I transform the above reactions usings JM109 cells I get no colonines on any of the plates, not even the single digest vectors. The only way I get colonies is if I only use uncut vector in the transformation. Also, I have tried blunt end cloning into the transfer vector pT7blue and that works fine. I get lots of white colonies and colony PCR has shown that they contain the insert. I don't think the problem is vector specific because I have tried to put the same insert into pEGFP-N1 and have exactly the same problem (ie no colonines even on the positive controls). Also the ligase definitely works because it is the same one I use for pT7blue cloning.



Does anyone have any suggestions on what could be going wrong? Could something be digesting away the cut ends of the vector so they cannot religate?


Is it possible that your antibiotics have gone bad or that your cells are not very competent? Being that your positive controls are not working it seems that it might be something simple like that.

-smu2-

QUOTE (smu2 @ Nov 26 2007, 07:29 AM)
QUOTE (ksmith @ Nov 23 2007, 03:44 AM)
I am trying to clone a 1.6 kb fragment into pcDNA3 expression vector (5.4 kb). I am cutting with 2 restriction enzymes, BamHI and XbaI.
When I run the gel of the restriction digests the position of the bands show that both enzymes are cutting the vector. I cut out the bands then use a QIAGEN gel extraction kit to extract the DNA.

My ligation reactions are:
1. pcDNA3 double digest only (negative control)
2. pcDNA3 double digest with insert double digest
3. pcDNA3 BamHI single digest only (positive control)
4. pcDNA3 XbaI single digest only (positive control)

When I transform the above reactions usings JM109 cells I get no colonines on any of the plates, not even the single digest vectors. The only way I get colonies is if I only use uncut vector in the transformation. Also, I have tried blunt end cloning into the transfer vector pT7blue and that works fine. I get lots of white colonies and colony PCR has shown that they contain the insert. I don't think the problem is vector specific because I have tried to put the same insert into pEGFP-N1 and have exactly the same problem (ie no colonines even on the positive controls). Also the ligase definitely works because it is the same one I use for pT7blue cloning.



Does anyone have any suggestions on what could be going wrong? Could something be digesting away the cut ends of the vector so they cannot religate?


Is it possible that your antibiotics have gone bad or that your cells are not very competent? Being that your positive controls are not working it seems that it might be something simple like that.


I think your transformation may be a problem. How you get the cells. Whether you centrifuge or not? If you are doing centrifuge, there may be a chance to cell death. Make sure about your transformation experiment.

-little-

QUOTE (phage434 @ Nov 23 2007, 09:54 AM)
Common problems: UV exposure of the DNA while imagining and cutting bands. Use long (365 nm) wave UV or blue light and limit exposure.

Poor transformation efficiency: Measure transformation efficiency of your cells and protocol. It should be at least 10**8 transformants/microgram of plasmid DNA.

Ligation concentrations: Using too high a concentration of vector and insert leads to concatamers rather than closed circular DNA. Concentrations should be < 20 ng/ul.

Ligation amounts: Use 1-3 ul of ligation to transform 50 ul of competent cells. More is inhibitory.

Outgrowth of cells: For tet and chloramphenicol resistance, use 1.5 - 2 hour outgrowth in nonselective medium rather than 1 hour to improve transformation efficiency. 1 hour works well for Kan and amp.



May I ask you one question? I use 40uL ligation system in 16oC overnight, insert : vector = 3:1 it is 120ng insert and 78ng vector in 40uL system. Does this fit into your <20ng/uL.

I am using 10uL ligation into 50uL competent cells. it is too much.

-newboy-

If you are getting no colonies, you need to approach this systematically.
1) how competent are your cells. Do serial dilutions of a known working plasmid dna sample and transform your cells with 2 ul of plasmid in 50 ul. Calculate the transformation efficiency of your cells in transformants/ug (not the best unit, but the traditional one). You need at least 10**7, preferably 10**8 or more.

2) Cut your plasmid with a restriction enzyme you plan on using (only one). Save some of the cut dna, religate another sample. Transform and verify that you have cut the plasmid (few transformants) and that you can religate (many transformants). Calculate your cutting/religation efficiency.

3) Same drill, other enzyme

4) Same drill, both enzymes. Religation should be lousy, especially if you gel purify the vector backbone.

5) Now, it will probably be obvious why your original reaction did not work. if not, then you have eliminated many of the potential problems and we can work from there.

-phage434-