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cloning from PCR


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21 replies to this topic

#16 Bego

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Posted 12 April 2010 - 10:08 PM

Hi!!
I am trying to ligate Il-8 and pIRES Ds Red,but I didnt succed.

I PCR IL-8 (which comes in a plasmid from Addegene).I designed primers with two different restriction sites,XhoI and Sac II.

I extracted IL-8 from the gel and digested with these 2 enzimes.I digested pIRES as well,with the 2 enzimes.

After ligation with DNA T4 ligase,and tranformation I didn`t get any colony.

I would like to know how I can precipitate my ligation before transformation.

And another question,should I use CAP?


Thanks in advance!!! :)

Edited by Bego, 12 April 2010 - 10:09 PM.


#17 William Parkar

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Posted 21 April 2010 - 10:14 PM

You could blunt end clone your fragment into pUC, then the digest should work. Remember that your vector should be de-phosphorylated and your insert phosphorylated.

http://www.flashpapers.com

#18 shane

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Posted 03 May 2010 - 10:31 PM

How far into your PCR goods are your limit sites? Enzymes commonly require not less than 6 bases distance from acknowledgement location to be present to cut. Also, manage you have a transformation control

#19 saeid ziaei

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Posted 27 June 2010 - 09:50 AM

You should check your ligation protocol and transformation.

#20 OA17

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Posted 06 September 2010 - 05:24 AM

I also think that cloning into a pUC or a TA vector will help excise your insert successfuly for the next cloning step.

I totally agree with Scott in that leaving only 6 bp at the end of the PCR fragment is ussually not enough for cloning, at least from my experience. Perhaps you could try to digest your insert for a longer time than the vector, because cutting a plasmid is much easier than cutting at the end of a PCR molecule (even more if you only have 6 bp at the end)and therefore the cutting eficiency of the insert may be lower than the vector.

Even though Xba is sensitive to Dam methylation, this should not be a problem for the PCR product, since it has not been introduced yet into a bacteria, so don't worry for that. This can be a problem for the vector if had not been proeviously transformed into a dam- strain.

Good luck!

#21 singularitygirl

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Posted 13 September 2010 - 05:16 AM

Hi,

I was wondering how you might know if your digests ar even ligations are any good at all? I agree with people that your insert is very small but I have seen it done so do not dispair! First of all, I was thinking that you have not said anything about setting up the proper controls. I like going back to basics if I have problems. For example, if I was to transform anything, I would of course set up to test competancy of your cells. Assuming that you have, for the transformations (with some antibiotic screening?) I would recommend setting up alot of controls so that you can pin point where the problem lies eg. vector + insert, insert only, undigested vector only, digested vector only, cells only all with antibiotic in addition to cells only with no antibiotic.

If no answers are here for direct cloning, then TA cloning (as someone mentioned above) is fantastic. It takes longer as it is 2 step but it works very well. You design your primers still with the restriction sites you want and PCR using both Vent polymerase (very good) and definately taq (which gives A&T overhangs) and you can directly ligate without digestion (TA eg. PCR2.1 TOPO is linear). Transform with all controls then what you can do is miniprep it up, restriction digest it to see if your insert is there. If so do a Midi, sequence it, blah blah, digest your TA clone with the primers of you choice, gel purify, ligate, transform etc etc.

The last thing, which I do not know much about is handling such small sizes of insert. I know a person or 2 who has done it and will ask. The thing is I am not sure if their insert was a result of PCR. Ill get back if I find something useful.

#22 Evanescence

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Posted 16 June 2011 - 07:55 AM

First, i agree with syracus_2000 and friends here...think we better always prepare the controls for the experiment then you will know what is wrong actually, probably because of the ligation product is too much, competent cells problem or problem ligation buffers/enzyme..the cloning maybe not successful due to fail to cut your RE site..




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