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annoying PCR cloning problem (season 2)


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#1 Biogareth

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Posted 09 April 2009 - 11:28 PM

Thank you all for advice!
Because I think the problem is that PCR products with insert cannot be cut well by restriction enzymes, later PCR products with insert was cloned into pGEM-T first, and then white-blue screen, I already got the plasmid with insert (checking by sequencing), afterwards cut the plasmid with NheI and PstI, the new plasmid has one NheI site and two PstI sites, but I think it can not be the problem to get the right insert, I already checked it on the gel.
As far as the digestion of vector was considered, I also check on the gel, it should be no problem.
After that, I do the ligation with different ratio of insert to vector (2:1. 3:1, 9:1), both overnight 4 C and room temper for 2 hours, but I just got one colony on the plate, no colony on control (without insert). When I checked this colony by colony PCR, there is still nothing, all the bands at the bottom of gel! It's so pity!
I already worked on that for 3 months! How come? ;)

Many thanks in advance!

Biograreth

#2 phage434

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Posted 10 April 2009 - 04:27 AM

So, be a lot more specific about how you are doing your ligation (reagents, amounts, temperatures, everything). Tell us about your competent cells (chemical, electroporation, strain, source, competence). How was your DNA prepared? Concentration of DNA? What is the destination plasmid?

If you have two PstI sites, does it matter which one is cut?

Are you certain your colony PCR works?

I would suspect your cell competence or transformation protocol, since you should be getting relatively high background transformation with uncut or partially cut vector in your negative conrol.

#3 Biogareth

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Posted 10 April 2009 - 06:12 AM

So, be a lot more specific about how you are doing your ligation (reagents, amounts, temperatures, everything). Tell us about your competent cells (chemical, electroporation, strain, source, competence). How was your DNA prepared? Concentration of DNA? What is the destination plasmid?

If you have two PstI sites, does it matter which one is cut?

Are you certain your colony PCR works?

I would suspect your cell competence or transformation protocol, since you should be getting relatively high background transformation with uncut or partially cut vector in your negative conrol.



I do the ligation reaction normorlly with 20 ul in total (when the ratio of insert: vector=9:1, 50 ul reaction solution was applied): 2 ul 10*ligation buffer (NEB), 1 ul T4 DNA ligase (NEB), the ratio of insert to vector is 2:1, 3:1, 9:1, the concentration of insert is 15.7 ng/ul, the concentration of vector is 7 ng/ul. Incubate them at 4C overnight, and also I try to incubate them at room temperature for 2 hours, afterwards inactivate at 65C for 20 min.
Ligation was transformed into XL10-gold competent cell with heat shock protocol, then transformants were plated on LB plates. The vector I used is a constitutive expression.
It doesn't matter which PstI site was cut. For colony PCR, it can not work.

#4 phage434

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Posted 10 April 2009 - 09:05 AM

When I asked about the colony PCR, I meant to ask if you any evidence the PCR reaction would work (with the primers, cycling conditions etc.) if the ligation was successful. Can you do a control reaction?

I don't immediately see anything wrong with your protocol. I would avoid heat killiing the ligase, which our experiments shows reduces transformation efficiency, but this should not be an issue here for such a simple cloning.

How are you preparing the DNA? Is it gel purified? If so, how do you visualize and cut it out? Do you have a gel image?

Is there some chance the DNA resulting from this is toxic to the cell? Are you adding an active promoter to a gene which is otherwise not highly expressed?

Can you do a control ligation at the same time? Cut your destination vector with a single enzyme, purify. Save some cut vector. Ligate another aliquot. Transform with uncut, cut but not ligated, and re-ligated vector. Count colonies.

What volume of DNA (insert and vector) go into the ligation? High volumes, composing a majority of the 20 ul reaction make the reaction sensitive to DNA contamination with inhibitors such as alcohol. Try working with higher DNA concentrations to reduce the potential contaminants in the ligation reaction.

#5 Biogareth

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Posted 14 April 2009 - 12:31 AM

When I asked about the colony PCR, I meant to ask if you any evidence the PCR reaction would work (with the primers, cycling conditions etc.) if the ligation was successful. Can you do a control reaction?

I don't immediately see anything wrong with your protocol. I would avoid heat killiing the ligase, which our experiments shows reduces transformation efficiency, but this should not be an issue here for such a simple cloning.

How are you preparing the DNA? Is it gel purified? If so, how do you visualize and cut it out? Do you have a gel image?

Is there some chance the DNA resulting from this is toxic to the cell? Are you adding an active promoter to a gene which is otherwise not highly expressed?

Can you do a control ligation at the same time? Cut your destination vector with a single enzyme, purify. Save some cut vector. Ligate another aliquot. Transform with uncut, cut but not ligated, and re-ligated vector. Count colonies.

What volume of DNA (insert and vector) go into the ligation? High volumes, composing a majority of the 20 ul reaction make the reaction sensitive to DNA contamination with inhibitors such as alcohol. Try working with higher DNA concentrations to reduce the potential contaminants in the ligation reaction.


Hi phage434,

I have repeated colony PCR three times, cannot work. However, when I do another PCR reaction, it can work.
As far as inactivating ligase was considered, ligation without inactivation was also used applied to be transformed into competent cells, but still can not work.
Insert and vector were purified by gel extraction kits, and were cut according to the gel image.
For the vector, there has an active promoter in it, and a guy in our team already successfully cloned his gene into this vector.
I do a control ligation, in which cut vector is added without insert, and there is no colony growing on the control plate.
Taking 2:1 ligation (insert: vector) as an example, 3 ul vector and 7 ul insert was added in 20 ul ligation reaction.

Thank you so much for your suggestion!

Biogareth

#6 phage434

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Posted 14 April 2009 - 04:57 PM

The usual problem with colony pcr is that people use far too many cells. Touch a tip to a colony, swirl into 50 ul of water, vortex the water, and then use 1 ul in your pcr reaction. Make sure you have a 10 minute or so 95C initial denaturing step to break the cells open. You need to get this working if you are going to use pcr to evaluate your clones. Do it with an already cloned vector to debug this.

You could be damaging the DNA by uv exposure during gel purification. Use long wave or blue illumination and minimize exposure times.

You need a control ligation which WORKS, not one which also fails. Cut your vector with a single enzyme, then religate. Transform both the cut and re-ligated samples to check for ligation and transformation efficiency.




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