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TOPO cloning problem


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#1 travelhappily

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Posted 13 May 2009 - 07:53 AM

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?

#2 bob1

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Posted 13 May 2009 - 04:16 PM

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.

#3 noelmathur

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Posted 13 May 2009 - 11:31 PM

I second bob1. In case if you have used proof reading polymerase, there is a protocol in TOPO manual on how to add -A overhang. It should work after that.
If your workflow was normal and still you had no colonies, don't ask why, you don't know what bugs do, just repeat, you may get it.

I am not a real fan of TOPO, I just prefer cut and paste, thats more reliable than TOPO. It takes time but its worth it.

#4 hanming86

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Posted 21 May 2009 - 03:39 AM

did u extend the final extension time to like 10 min. that 's required to add the extra A tail.
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#5 travelhappily

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Posted 21 May 2009 - 06:47 AM

But even if gene of interest didn't combind TOPO vector,the empty TOPO vector itself should combind itself and be transformed into TOP10 cell.Since TOPO vector has the amp resistance,they should grow on the LBamp100 plate after culturing over night.

Since according to lots of people's experience,you should see lots of colonies over night in TOPO cloning.

#6 almost a doctor

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Posted 21 May 2009 - 08:01 AM

TOPO vector 1ul
PCR product 4ul
salt solution 1ul
total 6 ul, room temperature let react for 30 min.



I've never used TOPO so not sure of protocol, but... arent you mising the Ligase in this reaction??? ;)

#7 travelhappily

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Posted 21 May 2009 - 08:50 AM

I've never used TOPO so not sure of protocol, but... arent you mising the Ligase in this reaction??? ;)


the ligase used in this experiment is called topoisomerase which has already existed in the vector solution.

#8 bob1

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Posted 21 May 2009 - 04:24 PM

But even if gene of interest didn't combind TOPO vector,the empty TOPO vector itself should combind itself and be transformed into TOP10 cell.Since TOPO vector has the amp resistance,they should grow on the LBamp100 plate after culturing over night.

Since according to lots of people's experience,you should see lots of colonies over night in TOPO cloning.


Actually, no - topo cloning precludes this possibilty, you should not get empty vector clones as the presence of the topoisomerases added to the ends of the vector cause steric hindrance, preventing self-ligation.

#9 almost a doctor

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Posted 22 May 2009 - 12:12 AM

I've never used TOPO so not sure of protocol, but... arent you mising the Ligase in this reaction??? :)


the ligase used in this experiment is called topoisomerase which has already existed in the vector solution.


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!

#10 travelhappily

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Posted 24 May 2009 - 06:48 AM

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!


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

#11 travelhappily

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Posted 24 May 2009 - 08:55 AM

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.

#12 jiajia1987

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Posted 24 May 2009 - 05:35 PM

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!


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.

#13 travelhappily

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Posted 25 May 2009 - 06:41 AM

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.


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!!!!!!!!!

#14 labmeiser

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Posted 27 May 2009 - 08:08 AM

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.




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?


Edited by labmeiser, 27 May 2009 - 08:11 AM.


#15 jiajia1987

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Posted 28 May 2009 - 05:20 PM

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!


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?




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