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Cloning PCR fragment


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#1 pm1234

pm1234

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Posted 10 May 2009 - 08:53 AM

Hi All,

I have never done molecular cloning before. I have to do the following and I would really appreciate if you could guide me with this.

-I am PCR amplifying a region (about 2kb) out from genomic DNA, which has two restriction sites.
- I am ligating this to another fragment (about 200 bp).
- I am then ligating the two fragments to a luciferase vector.

I am in the first step. I do a PCR reaction and with a Hi Fidelity Polymerase from Invitrogen, I get a pretty clean PCR product (most of the time just one band with one primer dimer). I've done this several times. I get a sharp band for my PCR product once in a while but lately I have been getting very faint bands. I set up 2-3 PCR rections and when doing the gel extraction, I combine them and elute with water (30 microliters). However, I get very very low yield (about 1- 9 ng/microliter). From my colleague, I found out that I will need atleast a microgram of DNA to start with. Please advise me if I can go ahead with the digestion at this point and what my setup should be and how much of stuff I should add. Any help is much appreciated.


Thanks,
P

#2 altobarn

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Posted 10 May 2009 - 11:12 AM

If you wish to increase the inserted fragment amount, perhaps you could consider to clone the PCR product into TA cloning vector subsequent by subcloning into your Luciferase vector.

You could get as much as you want from the cell culture, where you would transformed the TA vector+PCR fragment into competent cell and followed by miniprep.

-Altobarn-

Hi All,

I have never done molecular cloning before. I have to do the following and I would really appreciate if you could guide me with this.

-I am PCR amplifying a region (about 2kb) out from genomic DNA, which has two restriction sites.
- I am ligating this to another fragment (about 200 bp).
- I am then ligating the two fragments to a luciferase vector.

I am in the first step. I do a PCR reaction and with a Hi Fidelity Polymerase from Invitrogen, I get a pretty clean PCR product (most of the time just one band with one primer dimer). I've done this several times. I get a sharp band for my PCR product once in a while but lately I have been getting very faint bands. I set up 2-3 PCR rections and when doing the gel extraction, I combine them and elute with water (30 microliters). However, I get very very low yield (about 1- 9 ng/microliter). From my colleague, I found out that I will need atleast a microgram of DNA to start with. Please advise me if I can go ahead with the digestion at this point and what my setup should be and how much of stuff I should add. Any help is much appreciated.


Thanks,
P






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