Problem in cloning - (Nov/06/2007 )
Hi,
I have a problem in a simple subcloning experiment. I got 7 clones in the similar gene previously(mostly all my clones has insert). Usually, what i do i just amplify my interest gene with PCR include with RE sites EcoRI and Hind III, then double digest with RE, after gel purification i simply subclone with T4DNA liagase. For the past 2 months i am trying to clone similar gene (few less base pair) into the same vector, but it was not working. I try to use different ratio of vector and insert, different time of incubation (3-4 h, overnight at room temp, 4 C overnight) nothing worked. I got clone mostly all the time, it has only vector, no insert. I tried different company T4 DNA ligase, and RE.
If any one suggest something, It helps lots.
With regards,
Little
My suggestion is just verify your PCR primers carefully look for REsites whether the bases are sufficient for digestion or not. I too faced the same problem couple of months ago. Now i redisigned my cloning strategy by using vector based primers.
Hope this helps.
Hope this helps.
Ya i checked there is no problem for digestion, is there any possibility to affect the excess PCR product may interferes digestion?
Thanks for your suggestions
If u doubt that u can go for overnight digestion and check the possibility.
Based on your information that the restriction enzymes are fine, I would say there are two likely areas where you could be falling down. The first is your vector preparation. I'm not sure how you prepare your vector but in my opinion this is the single most important step in cloning. Here are the steps I do and everyone should do these:
-transform an aliquot of the vector into competent cells and plate O/N - vector stocks can sometimes readily degrade and when the quality of the vector stock is low the quality of the vector preparation can only be low - transforming the vector into competent cells allows new good quality vectors to be produced
-pick 3 x single colonies and grow them in as much culture as needed for a miniprep - single colonies are important because we only want a vector stock that is 100% quality - not a mixture of good vectors mixed in with deleted and crappy vectors
-miniprep the clones
-screen the clones at several positions in the vector to confirm that all parts of the vector are in tact - vectors like any cloned DNA get degraded and pick up deletions and screening the complete vector ensures that all parts of the vector are in tact and the vector is going to be functional - most of the time the screens are all good but occasionally they are not and that's when you appreciate the value of this process
-overdigest the vector with as many units of enzyme recommended for overdigestion - supercoiled plasmid DNA does not digest as efficiently as linear DNA and more units are required for complete digestion
-clean up the reaction using a reaction cleanup column or gel extraction column
-dephosphorylate the vector - even though you may double digest with non-compatible restriction enzymes, incomplete digestion leads to some singly-digested vectors that easily re-circularise in the ligation reaction
-electrophorese the vector to separate linear DNA from relaxed and supercoiled DNA - relaxed DNA (runs highest) is only nicked circular DNA = empty vectors, and supercoiled DNA (runs lowest) is completely undigested DNA = empty vectors. So although there should be very little of these two forms appearing on your gel, careful excision of the linear DNA reduces the number of empty vectors likely to cause background colonies. Singly-digested DNA will still be present but hopefully dephosphorylation removes most of that from the equation
So that's it. Excessive some may say but totally worth it if you want high efficiency ligations that produce minimal background colonies. I honestly believe people would have a lot less hassles with their cloning if they followed this procedure every time. I rarely get background colonies these days.
The second thing I would do is to perform some positive controls for the ligation. If you still have the previous vector and inserts you successfully cloned try these along side your current ligation and see how they work. That will tell you if it's the ligation reaction itself or the specific vector and insert that are causing you the problems.
Good luck,
Rob
-transform an aliquot of the vector into competent cells and plate O/N - vector stocks can sometimes readily degrade and when the quality of the vector stock is low the quality of the vector preparation can only be low - transforming the vector into competent cells allows new good quality vectors to be produced
-pick 3 x single colonies and grow them in as much culture as needed for a miniprep - single colonies are important because we only want a vector stock that is 100% quality - not a mixture of good vectors mixed in with deleted and crappy vectors
-miniprep the clones
-screen the clones at several positions in the vector to confirm that all parts of the vector are in tact - vectors like any cloned DNA get degraded and pick up deletions and screening the complete vector ensures that all parts of the vector are in tact and the vector is going to be functional - most of the time the screens are all good but occasionally they are not and that's when you appreciate the value of this process
-overdigest the vector with as many units of enzyme recommended for overdigestion - supercoiled plasmid DNA does not digest as efficiently as linear DNA and more units are required for complete digestion
-clean up the reaction using a reaction cleanup column or gel extraction column
-dephosphorylate the vector - even though you may double digest with non-compatible restriction enzymes, incomplete digestion leads to some singly-digested vectors that easily re-circularise in the ligation reaction
-electrophorese the vector to separate linear DNA from relaxed and supercoiled DNA - relaxed DNA (runs highest) is only nicked circular DNA = empty vectors, and supercoiled DNA (runs lowest) is completely undigested DNA = empty vectors. So although there should be very little of these two forms appearing on your gel, careful excision of the linear DNA reduces the number of empty vectors likely to cause background colonies. Singly-digested DNA will still be present but hopefully dephosphorylation removes most of that from the equation
So that's it. Excessive some may say but totally worth it if you want high efficiency ligations that produce minimal background colonies. I honestly believe people would have a lot less hassles with their cloning if they followed this procedure every time. I rarely get background colonies these days.
The second thing I would do is to perform some positive controls for the ligation. If you still have the previous vector and inserts you successfully cloned try these along side your current ligation and see how they work. That will tell you if it's the ligation reaction itself or the specific vector and insert that are causing you the problems.
Good luck,
Rob
Hi Rob,
Thanks lot for giving very impressive and valuable ideas. I highly appreciate your thoughts and reply. I try to follow all your comments and reply to you once i got some good results. Yesterday, i got some interesting results, i checked the vector quality with a previously prepared insert, i got 50% positive clones. So, I think your point may be right, the vector is not in good quality or not completely digested, or the new insert is not completely digested.
Thanks for yours wishes
little
Hope this helps.
Ya i checked there is no problem for digestion, is there any possibility to affect the excess PCR product may interferes digestion?
Thanks for your suggestions
Over digestion would kill sites and make them impossible to clone. So watch out if you over digested. Which enzymes are you using ?
Hope this helps.
Ya i checked there is no problem for digestion, is there any possibility to affect the excess PCR product may interferes digestion?
Thanks for your suggestions
Over digestion would kill sites and make them impossible to clone. So watch out if you over digested. Which enzymes are you using ?
I tried both Promega and NEBS
Hope this helps.
Ya i checked there is no problem for digestion, is there any possibility to affect the excess PCR product may interferes digestion?
Thanks for your suggestions
Over digestion would kill sites and make them impossible to clone. So watch out if you over digested. Which enzymes are you using ?
I tried both Promega and NEBS
I would add something to it. Make two vector digestions parallel. and Check an aliquot from one of them on gel after a couple of hours if it is completely digested. You can use the other one for your ligations. That saves the sample and you dont have to worry about overdgestion by keeping at overnight.