Electrophoresis, pls urgent - (Jun/13/2007 )
Hello perneseblue, you are doing a great job....gave me such a brilliant idea.....
I checked my sequences again and found one restriction site (ClaI) which is only present in the fragment which I want to digest out and it is not present in the fragment which I am cloning into. So I am going to elute out single cut NcoI fragment and the partial digested BamHI-NcoI fragment together and setting up a ligation with PciI-BamHI cut PCR fragment. After overnight ligation I will digest the all ligation mixture with ClaI and going to use for transformation.... I red this digestion step somewhere in this forum posted by you???? but unable to trace out it by now...Could you please just give me the link to that post or could you please explain it briefly once again ???????????Thank you very much.....
transform your DNA mixture in bugs.. n then do the colony PCR to recognise ...which bug is having what DNA.. grow the bug and do the maxi.. enjoy..
but a simple problem here.. if you got both DNA in same bug..no worries..
test the same bug with both plasmid primers.. and select the colony which is single positive only..
what do you think??? i think its possible and very easy..
The plan is possible but I don't think it will be easy.
The linearised NcoI (9.23kb) vector will very likely religate more readily, then the partial digest vector (BamHI/NcoI) will ligate to the new insert. So unless the linearised NcoI vector can be removed or decreased in some way, the number of vector+new insert colonies will be vastly outnumbered by the religated vector. Colony Southernblot will definately resolve this or a large colony PCR (you will definately need multichannel pipettes to do this). I would screen ~200+ colonies
However, if the new insert can kill the NcoI site. An NcoI digest after ligation will fix this problem. FatI. PciI digest? Nope?
If the partial digest bands can not be resolved, and the strategy suggested by LaboratoryHD doesn't work, you could monkey around with the vector.
Assuming that your vector does not have a BglII site.
You could ligate the NcoI cut vector with an oligo which carries a BglII site. Importantly one of the primers does not reform the NcoI site. It kills the site. While the second primer keeps the site.
Orientation of the insert has to be done by PCR. One of the primers used to make the site and one primer binding to the vector.
Finally,another alternative would be to avoid the problem and go hunt for an empty vector.
yes you can mix both agarose, just remember that methaphor need to be hidrate for 10 min, add the regular and when heating in microwave do it slowly. methaphor is good for separete fragments that are very near, but is very expensive and hard to work wiith . The regular one is good to separete big fragments and easy to use, so when combine you get the best of each.I had use this combination many times for difficult to separate fragments and never have problems.
Thank you, learned something really new and useful......
That is quite a nice plan.
In fact you can actually use a NcoI-BamHI PCR fragment. No need to use PcI.
- elute out single cut NcoI vector and the partial digested BamHI-NcoI vector together
- ligate with NcoI-BamHI PCR insert
- take the ligation mix, phenol-chloroform to deactivate ligase.
- move supernatant to new tube, and percipitate DNA with Ethanol and NaAcetate. Best if you do this in a small 0.5ml tube. The small tube makes it easier to see the pellet. Also adding dextran will help make the pellet easier to see.
-wash with 70% ethanol. Spin to make sure the pellet hasn't fallen off the side of the tube.
- after that resuspend DNA in appropriate buffer for a ClaI digest. Cutting any religated NcoI cut vector
-after digest, percipitate DNA, wash with 70% EtOH (spin), and resupend in sterile distilled water. And the DNA should be ready for transformation.
Thank you very much perneseblue, there is a NcoI restriction site inside my PCR fragment. That is the reason I used PcIi...Any way thank you very much for your help..........
In fact you can actually use a NcoI-BamHI PCR fragment. No need to use PcI.
- elute out single cut NcoI vector and the partial digested BamHI-NcoI vector together
- ligate with NcoI-BamHI PCR insert
- take the ligation mix, phenol-chloroform to deactivate ligase.
- move supernatant to new tube, and percipitate DNA with Ethanol and NaAcetate. Best if you do this in a small 0.5ml tube. The small tube makes it easier to see the pellet. Also adding dextran will help make the pellet easier to see.
-wash with 70% ethanol. Spin to make sure the pellet hasn't fallen off the side of the tube.
- after that resuspend DNA in appropriate buffer for a ClaI digest. Cutting any religated NcoI cut vector
-after digest, percipitate DNA, wash with 70% EtOH (spin), and resupend in sterile distilled water. And the DNA should be ready for transformation.
Let me see if I understand your problem. You have a vector like this (-----N----B1------B2-------)
You want to insert a DNA ( N------B ) into (----N----B1----)
If I were you, I would insert ( N------B ) into (----N-----------B2----) first, and then put the B1------B2 back.
Thank you WHR, its not a bad idea but I almost suceeded with partial digestion method, but I would definitly keep your idea in mind for my next cloning........thank you..
You want to insert a DNA ( N------B ) into (----N----B1----)
If I were you, I would insert ( N------B ) into (----N-----------B2----) first, and then put the B1------B2 back.
though keep in mind that B1-----B2 might contain important bits of the plasmid (eg origin of replication, antibiotic marker). So keep a good eye on the plasmid's structure if you want to start monkeying with the plasmid's backbone.