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cloning PCR fragment

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2 replies to this topic

#1 jonathannimal



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Posted 01 September 2004 - 01:09 AM

I have amplified a PCR fragment. The molecular size of the fragment is 3.5KB. The fragment has restriction site on both the ends. the restriction site is SalI & BamHI. Restriction enzymes works well. My ligation enzyme also works well in the control. But I am not able to get the clone. No colonies appear in the plate.
Please help me

#2 phegene



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Posted 01 September 2004 - 09:25 AM

I normally gel extract PCR products with Quiagen's gel extraction kit, then clone that into my chosen vector. Since it is a very long fragment, make sure to add correct molar amounts of your fragment and vector in the ligation. Also, how much of your ligation are you transforming... certain ecoli cells are sensitive to DNA amounts transformed. And if this is just a low efficiency problem, make sure to plate everything that you transformed. Good luck!

#3 tfitzwater



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Posted 01 September 2004 - 01:45 PM

Sal I is notorious for not working very well when there are only a few basepairs upstream. This is one of the more frequent complaints about cloning PCR products that appear in the numerous bio forums on-line. See the appendix in the back of the New England Biolabs catalog.

To check the restriction efficiency, use T4 kinase to label the 5' ends with 32P. Then cut one aliquot with Sal I, one aliquot with BamH I, and a third aliquot with both enzymes. Run the products on a gel with uncut labeled DNA and quantify the amount of label remaining on the insert.

Option 1: get a TA cloning kit and forget about reestriction cuts.
Option 2: polish the ends and then cut with BamH I. Ligate into vector cut with BamH I and a blunt-end restriction enyme.

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