TOPO cloning problem - (May/13/2009 )
bob1 on May 13 2009, 04:16 PM said:
What polymerase did you use? If you used a proof reading one, they have an exonuclease capability that means that they don't leave A overhangs on the end of the PCR product - something Topo TA cloning relies on.
I use GoTaq DNA polymerase from Promega.
I do think they are not proof reading one,since all my lab members did well in TOPO cloning using GoTaq DNA polymerase.
-travelhappily-
travelhappily on May 24 2009, 10:48 PM said:
almost a doctor on May 22 2009, 01:12 AM said:
Didn't know that, cool!
In that case I do agree with bob1 and co. make sure you A-tail your PCR product. If you are using a proofreading polymerase, all you have to do at the end of your PCR is add extra dATP and Taq and icubate at 72C for 15-20min.
Good luck!
In that case I do agree with bob1 and co. make sure you A-tail your PCR product. If you are using a proofreading polymerase, all you have to do at the end of your PCR is add extra dATP and Taq and icubate at 72C for 15-20min.
Good luck!
Thanks so much!!!
I will try to elongate for 10min since I used 5 min in elongation before.
By the way,how carefully should I treat TOP10 cell to make sure the efficiency of the cells?
They are so ......weak!!!!!!! Make me crazy!!!1
I don't know what do you mean by treat TOP10 cells.
But what I usually do is to thaw them on ice and after adding in the ligated products, i simply use the pipette tip to swirl the contents ONCE. that's it. never pipette them up and down. the cells can die. It will be better if you clean up the PCR products before doing the ligation too.
-jiajia1987-
jiajia1987 on May 24 2009, 05:35 PM said:
I don't know what do you mean by treat TOP10 cells.
But what I usually do is to thaw them on ice and after adding in the ligated products, i simply use the pipette tip to swirl the contents ONCE. that's it. never pipette them up and down. the cells can die. It will be better if you clean up the PCR products before doing the ligation too.
But what I usually do is to thaw them on ice and after adding in the ligated products, i simply use the pipette tip to swirl the contents ONCE. that's it. never pipette them up and down. the cells can die. It will be better if you clean up the PCR products before doing the ligation too.
The moment I took them out from -80freezer,I put them into ice---all tube under ice!!
I never pipette them up and down,I even didn't touch them untill I tried to open the lid and flick very lightly to make sure whether the cells have thawed.And I am very sure I didn't let them stay in the ice for more than 3-4 min because I know we need to use them immediately when they have thawed.
I even cut the tips to make a bigger opening so that I might not hurt cells if I pippette them out from 2ml tube to 1.5 ml tube for transformation.(here is the only one step we need to pipette them since they are stored in 2ml tube but heat shock machine can only accept 1.5ml tube.And I tried this step before and got good result of transformation.)
I didn't get very good result of pUC19 transformation ---the efficiency is low.I didn't get colonies as much as normal condition should be.So I guess I should really have transformation efficiency problem.
But I really treat TOP10 cell very carefully to make sure I may not hurt them.I am gonna CRAZY!!!!!!!!!
-travelhappily-
Did the TOP10 cells come in a kit? Last time I used the Champion dTOPO directional cloning kit from Invitrogen the TOP10 cells were contaminated. I switched to the DHEs we normally use for plasmid prep and it worked fine (just in case you are using this kit). But I guess that doesn't really help since your puc19 plate worked. Sorry.
travelhappily on May 13 2009, 10:53 AM said:
I did TOPO cloning yesterday and spread the bacteria onto LB ampicillin 50 ug/ml and culture at 37 over night.
BUT NOTHING grows out!!!!
TOPO vector 1ul
PCR product 4ul
salt solution 1ul
total 6 ul, room temperature let react for 30 min.
then I did the normal transformation into top10 cell.
and I did one positive control--pUC19.
and grow them onto LB plate containing ampicillin 50ug/ml.
culture plates at 37 over night.
Nothing on the TOPO vector & PCR product.
pUC19 plate grow fine.there are lots of colonies on pUC19 plate.
WHY?
BUT NOTHING grows out!!!!
TOPO vector 1ul
PCR product 4ul
salt solution 1ul
total 6 ul, room temperature let react for 30 min.
then I did the normal transformation into top10 cell.
and I did one positive control--pUC19.
and grow them onto LB plate containing ampicillin 50ug/ml.
culture plates at 37 over night.
Nothing on the TOPO vector & PCR product.
pUC19 plate grow fine.there are lots of colonies on pUC19 plate.
WHY?
-labmeiser-
jiajia1987 on May 25 2009, 09:35 AM said:
travelhappily on May 24 2009, 10:48 PM said:
almost a doctor on May 22 2009, 01:12 AM said:
Didn't know that, cool!
In that case I do agree with bob1 and co. make sure you A-tail your PCR product. If you are using a proofreading polymerase, all you have to do at the end of your PCR is add extra dATP and Taq and icubate at 72C for 15-20min.
Good luck!
In that case I do agree with bob1 and co. make sure you A-tail your PCR product. If you are using a proofreading polymerase, all you have to do at the end of your PCR is add extra dATP and Taq and icubate at 72C for 15-20min.
Good luck!
Thanks so much!!!
I will try to elongate for 10min since I used 5 min in elongation before.
By the way,how carefully should I treat TOP10 cell to make sure the efficiency of the cells?
They are so ......weak!!!!!!! Make me crazy!!!1
I don't know what do you mean by treat TOP10 cells.
But what I usually do is to thaw them on ice and after adding in the ligated products, i simply use the pipette tip to swirl the contents ONCE. that's it. never pipette them up and down. the cells can die. It will be better if you clean up the PCR products before doing the ligation too.
Now I am confused. Do you mean TOP10 cells provided along with the TOPO cloning kit? or TOPO10 cells prepared by you? don't you use the TOPO vector provided?
-jiajia1987-
I suspect there's somehting wrong with he TOPO reaction or the lack of A-tail. TOP10 cell is pretty robust. and u can easily make them chemically or electrocompetent. since the control is working, that means the cells are ok. they can take abit of "less-gentle" treatment. either that or ur clone is toxic to the cell.
-hanming86-