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PCR and cloning - (Feb/18/2009 )

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predoc on Feb 21 2009, 02:45 PM said:

Hi pernese blue,

I ran the ligation mix this morning. I also ran uncut starting vector that has a size of 8.5 kb along with it. The new vector size should be 10.5 kb. I saw that the supercoiled starting vector is about the same size as the band I see for the ligated DNA. Do you think I have the insert in my vector?

Thanks



if both the uncut plasmid of both the ligated and starting vector run at the same level, then highly unlikely that the ligation worked.

I somehow have a feeling that the DNA is not getting digested properly. Is there possibility to use any other enzyme? Try to go over the sites.

What is your ligation recipe? How much DNA do you use? Also could you verify if the restriction enzymes are working optimally.

-scolix-

Hi Scolic,

My ligation recipe has about 120ng of digested vector and about 500 ng of digested insert(obtained from PCR). I ligate a total reaction of 20ul. I add 1 ul of T4 DNA ligase. The ligation is o/n at 4C.

Thanks a lot

Predoc....

scolix on Feb 21 2009, 07:06 AM said:

predoc on Feb 21 2009, 02:45 PM said:

Hi pernese blue,

I ran the ligation mix this morning. I also ran uncut starting vector that has a size of 8.5 kb along with it. The new vector size should be 10.5 kb. I saw that the supercoiled starting vector is about the same size as the band I see for the ligated DNA. Do you think I have the insert in my vector?

Thanks



if both the uncut plasmid of both the ligated and starting vector run at the same level, then highly unlikely that the ligation worked.

I somehow have a feeling that the DNA is not getting digested properly. Is there possibility to use any other enzyme? Try to go over the sites.

What is your ligation recipe? How much DNA do you use? Also could you verify if the restriction enzymes are working optimally.

-predoc-

Hi PCR boy, I spread the transformation reaction in 2 titers. The first one is 50ul of a 1050ul transformation reaction. On this plate I typically get 5-10 clones. In the second, I spin down all the remaining cells and plate all of them in 50ul. On this plate I get more than 20 clones. So I pick 10 at first. Check them and then pick 10 more from either plate. In all I have checked 20-25 each time I did this. I did the negative control only once when I started in Jan with the first technique of introducing the tag by PCR. I did it again yesterday. i will check my latest result and tell you in about an hour.

Thanks,
predoc


pcrboy on Feb 20 2009, 11:49 PM said:

I think the fact you have successfully cloned other vectors, suggests that your reagents are fine. I also doubt it is your ligation since its an overnight reaction. Also, I disagree with an earlier post that screening 20 clones is too small. How many clones are you getting from your ligation transformation? Do you have a transformation with vector-only for a negative control?

-predoc-

hi
i also faced the same problem while cloning PCR product directly.
Then I got an idea of first cloning that PCR product in any T/A cloning vector. Then from that you just go for digestion of insert.
I also suggest you to gel purify ur vector and insert.

Hope it'll work

-PMB_stu-

I do have colonies on my negative control plate but when I plate the concentrated cell mix. Unfortunately, my ligation mix also gives me about the same number of colonies, more on the conc cells. Do you think EcoRV and XhoI need more digestion than 4-6 hours??

Thanks

predoc on Feb 22 2009, 06:18 AM said:

Hi PCR boy, I spread the transformation reaction in 2 titers. The first one is 50ul of a 1050ul transformation reaction. On this plate I typically get 5-10 clones. In the second, I spin down all the remaining cells and plate all of them in 50ul. On this plate I get more than 20 clones. So I pick 10 at first. Check them and then pick 10 more from either plate. In all I have checked 20-25 each time I did this. I did the negative control only once when I started in Jan with the first technique of introducing the tag by PCR. I did it again yesterday. i will check my latest result and tell you in about an hour.

Thanks,
predoc


pcrboy on Feb 20 2009, 11:49 PM said:

I think the fact you have successfully cloned other vectors, suggests that your reagents are fine. I also doubt it is your ligation since its an overnight reaction. Also, I disagree with an earlier post that screening 20 clones is too small. How many clones are you getting from your ligation transformation? Do you have a transformation with vector-only for a negative control?

-predoc-
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