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Cloning Problems! Need Help!


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#1 Gam8Doc

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Posted 21 May 2009 - 08:33 AM

I switched to a lentiviral plasmid that has the compatible restriction sites of my insert. I have done 2 ligations in both DH5a and Top10 bacteria and both times got no colonies to grow on my plates.

Details:
-Insert is 2.7kb (I did a double digest and ran the dna on a gel I get a 3kb and 2.7kb fragment, both bands are separated enough to cut out the 2.7kb band (as far as I can tell) and purify with the Qiagen Gel extraction kit).
-Vector is 7.3kb and I am only removing 30bp when I do a double digest in the multiple cloning region to open up the plasmid. Again I run it on a gel and cut out the band and purify.
-Ligation: I do a 1:3 and 1:5 vector:insert molar ratio (I get the concentration from a Nanodrop). I use T4 DNA ligase from Invitrogen and I have tried the ligation at room temperature for 1h and at 14C for ~20h before bacterial transformation.
-My DNA fragment concentrations are rather low (6.43ng/ul for the vector and 11.11ng/ul for the insert) and after the ligation I dilute the ligation reaction 5x in H2O before bacterial transformation (as per Invitrogens recomendation on the protocol for T4 Ligase).

Anyone have any suggestions? Some of my questions are 1) how do I know that I am only removing the 2.7kb fragment? Is there another way to purify this fragment rather than running it on a gel and extracting the band? and 2) is there anyway to improve my ligation efficiency? Can anyone see anything else that might be causing me trouble?

Thanks!

#2 phage434

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Posted 21 May 2009 - 01:35 PM

Run a control transformation to make sure your cells are competent, and that your transformation technique is good. I'd recommend transforming 1ul of a serial dilution of pUC19 plasmid in TE at 1 ng/ul, 100 pg/ul and 10 pg/ul. You should achieve at least 1e8 transformants/ug of DNA.

It is very unlikely that no colonies result from this experiment, unless your transformations are failing seriously. The more likely failure would be no inserts or very high background.

Do you have the right antibiotic?

#3 Vini

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Posted 22 May 2009 - 01:34 AM

Run a control transformation to make sure your cells are competent, and that your transformation technique is good. I'd recommend transforming 1ul of a serial dilution of pUC19 plasmid in TE at 1 ng/ul, 100 pg/ul and 10 pg/ul. You should achieve at least 1e8 transformants/ug of DNA.

It is very unlikely that no colonies result from this experiment, unless your transformations are failing seriously. The more likely failure would be no inserts or very high background.

Do you have the right antibiotic?


i suspect ur ligations are not occuring properly. keeping the reactiona at 14C for 20 hrs is no help. I mean u either get it in 6-9 hrs. (at max.) or not at all.

#4 Gam8Doc

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Posted 22 May 2009 - 09:16 AM

Run a control transformation to make sure your cells are competent, and that your transformation technique is good. I'd recommend transforming 1ul of a serial dilution of pUC19 plasmid in TE at 1 ng/ul, 100 pg/ul and 10 pg/ul. You should achieve at least 1e8 transformants/ug of DNA.

It is very unlikely that no colonies result from this experiment, unless your transformations are failing seriously. The more likely failure would be no inserts or very high background.

Do you have the right antibiotic?


i suspect ur ligations are not occuring properly. keeping the reactiona at 14C for 20 hrs is no help. I mean u either get it in 6-9 hrs. (at max.) or not at all.


I have done the control plasmids. I used pUC19 which is resistant to Amp. I cultured these cells on Amp plates (high # of colonies) and on Kanamycin plates (No Colonies). Kanamycin is the antibiotic that my plasmid is resistant too. I also was able to transform and grow the un-cut empty vector plasmid on the kanamycin plates with no problems. I am only having problems when I try the ligations and see no colonies.




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