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ligation

double digestion ligation transformation

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8 replies to this topic

#1 student47

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Posted 23 August 2013 - 12:38 AM

I double digested using xho1 and sal1 and ligated it wit t4 ligase overnight at 4 deg C, had a vector only control too. then transformed with heat shock into d5 alpha competent cells. now on the plates i have many colonies (not doing blue white selection so all are just normal white colonies) on the control as well as others. i haven't checked on gel yet but seems like i have a lot of background , either uncut vectors or religated vector. i checked the cut sites for these two enzymes, the both give a 5 bp overhang which are very similar with only one base pair difference, that is CAGTC and GAGTC. can they still religate themselves?? if yes, is there a way to do this without changing it into new enzymes which will require starting from primers? thanks in anticipation.



#2 susnata

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Posted 23 August 2013 - 01:06 AM

i am not sure if i understood your problem clearly..

 

but both Sal1 and Xho1 give a 4 bases overhang and they are exactly the same..

 

what is the plasmid that you are using.. n is there a selectable marker where you are ligating your insert in the vector?

 

if you have an antibiotic marker for instance, it should not be much of a problem to screen for your clones.



#3 susnata

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Posted 23 August 2013 - 01:29 AM

n i guess ligation using  T4 ligase should be kept at 16^C



#4 student47

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Posted 23 August 2013 - 02:08 AM

hi it was not a t4 ligase and temperature was supposed to be 4 deg c i confirmed. the plasmid has ampicillin reistance gene but the insert i am trying to ligate into the plasmid is just a 500 bp pcr gene product. whic i cleaned and then double digested with xho1 and sal 1. in a double digestion you prefer 2 different enzymes so that the overhangs are different and the insert to go in the right way in, in this case i did not check what the overhangs were,  but as you said the overhangs are same, my question is

 

 

is it possible the double digested product from two different enzymes (which i just found out is unfortunately) giving same/similar overhangs get religated themselves to give the circular plasmid?   



#5 almost a doctor

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Posted 23 August 2013 - 02:18 AM

is it possible the double digested product from two different enzymes (which i just found out is unfortunately) giving same/similar overhangs get religated themselves to give the circular plasmid?   

 

The short answer is YES.   It doesn't matter how the overhangs got generated, if the ends are compatible (as in this case are, in fact the same), they will ligate.  

 

I'd strongly suggest you redesign your experiment and choose different enzymes with non-cohesive ends.   "starting from primers" really is your best (even only) option here.   Also, with your current design you'll need to screen for correct direction of your insert.  



#6 student47

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Posted 23 August 2013 - 04:06 AM

thank you..



#7 phage434

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Posted 23 August 2013 - 10:00 AM

Also, avoid SalI as an enzyme when planning on cutting at the end of PCR fragments. It's a known trouble maker.



#8 labtastic

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Posted 05 September 2013 - 04:35 AM

Consider doing a quick colony pcr on some of your colonies to see if any have the insert. It takes very little set up time and minimal reagents, and will tell you definitively which colonies, if any, have the insert. Though it won't tell you if it's the right direction or not. You will probably have to sequence the ones with the insert to confirm directionality.

 

Or...use different cut sites as others have suggested.



#9 phage434

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Posted 05 September 2013 - 06:19 AM

Colony PCR is not definitive, since the PCR sometimes fails on good constructs, and sometimes succeeds (due to DNA on the plate) with bad ones. It's far better than nothing, however. It can be directional if used with one primer on the vector and a second on the insert. You usually want to be doing ligations with different overhangs at each end, to avoid the directionality issue, in any case.







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