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ligation of PCR product

About microscopic forms of life, including Bacteria, Archea, protozoans, algae and fungi. Topics relating to viruses, viroids and prions also belong here.

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ligation of PCR product

Postby biology_06er » Wed Oct 05, 2011 8:01 am

Hi there,

Today I used my PCR product (that was cleaned up) to ligate into PET expression...just want to see if I went about it the right way:

Cut my vector with xho1/ecor1 and corresponding buffer and water up to a volume of 20uL...kept at 37 degrees for around an hour
cut my pcr product with same enzymes/buffer/water up to a volume of 20uL and kept at 37 degrees for an hour

ran a gel to see if dna was present..it was so then took

7uL of insert
1uL of vector
1uL ligase
1uL buffer

and ran one without insert (substituted with water)...

was that right? in parallel I also did a gel purification exp and ligated with simliar amounts...but just want to know if the pcr product procedure I did was correct?

Thanks!
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Postby canalon » Wed Oct 05, 2011 1:13 pm

I would have used the purified (gel or column) DNA rather than the restriction mix for the ligation. The salts and whatever in your restriction buffer might not make your ligase happy and you have not even diluted them (80% of your reaction is basically in the restriction buffer). I would also have dephosphorylated the vector to reduce the probability of religation of partially digested vectors (cut by only one enzyme).
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Re: ligation of PCR product

Postby biology_06er » Wed Oct 05, 2011 11:15 pm

I did also use the gel purified DNA but also was told to try ligating the pcr product straight into the vecotr...i have no idea what the "procedure" for this is..i have looked and looked for one online...was this part at least correct?

Cut my vector with 1uLxho1 and 1uL ecor1 and corresponding buffer (2uL as were compatiable) and water (11uL) kept at 37 degrees for around an hour
cut my pcr product with same enzymes/buffer/water up to a volume of 20uL and kept at 37 degrees for an hour

this to be seeems right its the next step im not sure about (however this could be wrong to)..do i run all 20 on a gel and cut out and gel purify
or do i clean up dna (phenol/cholorform extraction) and use the dna i get from that?

and deterimine ratios by running a gel etc
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Re: ligation of PCR product

Postby biology_06er » Thu Oct 06, 2011 2:01 am

n/m...I understand now!
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