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help wih the ligation


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7 replies to this topic

#1 roshanbernard

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Posted 15 June 2009 - 06:01 PM

hi, i am clonning a synthetic gene which is 32 bp long with restriction site i.e EcoRv and SalI...For EcoRV cut blunt ligation it says we need to incubate for 16 hours in room temperature and SalI cut sticky end says we need to incubate at 4 degree for 16 hours.....

i am using roche T4 ligase...and wanted some help to know how to go about ligation.needed to know how can i ligate when i use both blunt and sticky end ligation.....

#2 erne

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Posted 16 June 2009 - 12:12 PM

16 degree for 16 hrs and 4 degree for 16 hrs, works for every ligation. Make sure your ligation buffer is new/fresh.

#3 mastermi

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Posted 16 June 2009 - 12:21 PM

Can't you use klenow to make both ends blunt?

#4 roshanbernard

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Posted 16 June 2009 - 06:15 PM

Thank you erne and mastermi for the suggestion....well,first i will try going ahead with the ligation for 16 hour..if this does not work then i am planning to carry upon with the klenow fragment addition...

#5 roshanbernard

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Posted 24 June 2009 - 10:53 PM

when i tried doind the ligation of 7.3 kb vector cut with the EcoRV(blunt end) and sal1(sticky end) ans my synthetic gene cut with the same restrction enzyme......i got the colonies in the plate....hen i extracted the plasmid the size of the plasmids were 5 kb...what might be the reason..can it be contamination???
i had prepared quite a few samples....4 degree over night..and other sample at 25 degree and other one at 4 degree for 5 hours then shift that sample to 25 degrees for 11 hours....
but in all samples i got the same 5kb plasmid containing colonies....
i am totally confused,thinking why its happening with me....can anyone help me out please...

#6 angad

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Posted 07 July 2009 - 12:04 PM

Please help:

I am trying to clone an insert 1.5Kb into a vector 7 Kb.

I started by amplifying the insert and first cloning into the TOPO vector. That part was easy. Now I am trying to cut this insert out of TOPO vector and pasting in the new vector (7Kb).

I can see the bands on the gel after double digest (NotI and XbaI), I cut them out-the insert and the vector arms and purify with qiagen. My yield drops a lot using this kit but I still have enough DNA to do the ligations. I have tried equimolar ratios and 1:3 ratio of V:I. I get no colonies.

Controls for transformation work fine, ligation controls have also worked although not tonnes of colonies.

At this point I am frustrated and need help.

What may be going wrong?

Please anyone???

#7 phage434

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Posted 08 July 2009 - 05:19 AM

Trashing your DNA with UV is the most likely problem. Use long wave UV or better blue to visualize your bands. Some people visualize one fragment, mark it, and use the marks to cut an unexposed gel fragment. Adding guanine to your gel is supposed to minimize the problems -- I have not tried this.

You could also be doing the ligations at too high a concentration. With an 8Kb desired circular product, you want low concentrations for the ligation. Around 1 ng/ul in the final ligation would be good.

Check the competence of your cells. 10^7 cfu/ug or greater is probably important.

This should be easy, unless your construct is toxic.

#8 angad

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Posted 08 July 2009 - 06:32 AM

Trashing your DNA with UV is the most likely problem. Use long wave UV or better blue to visualize your bands. Some people visualize one fragment, mark it, and use the marks to cut an unexposed gel fragment. Adding guanine to your gel is supposed to minimize the problems -- I have not tried this.

You could also be doing the ligations at too high a concentration. With an 8Kb desired circular product, you want low concentrations for the ligation. Around 1 ng/ul in the final ligation would be good.

Check the competence of your cells. 10^7 cfu/ug or greater is probably important.

This should be easy, unless your construct is toxic.



Thanks!

I have done a similar size ligation in the past and hence have been using similar molar ratios.

I will try what you say-marking with the standard and cutting it without exposing.

My competent cells are really fine and give me good transformation efficiencies.




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