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TOPO TA cloning problem


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#1 molstudent

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Posted 02 April 2010 - 02:31 PM

Hello,
I've been having problems cloning with the invitrogen TOPO TA cloning kit. I've read many topics on this forum but none seem to have helped me. I've used this kit many times before and it worked wonderfully but for some reason it isn't now.

When I perform the TOPO clone reaction, my plates originally gave many white colonies and few blue but they were negatives. I concluded there must be smaller fragments from my PCR reaction that were getting in the topo vector so I tried first gel purifying my fragments before topo and this gave me many blue colonies but few white colonies. however, these few white colonies sometimes contained the fragments i wanted but for some reason they were very messed up at the ends mainly and i dont know why they are messed up or why i get so few white colonies.

I have tried elongase and taq for my polymerases and they both give me the same results. i also performed the control PCR that comes with the topo kit and it worked very well, much like i'm use to. could it be that my primers are messed up since i'm seeing problems at the ends of the fragments i want? and could that also be why i don't get very many white colonies since the "A" on the ends of the strands is important for the TOPO TA kit? I've been having problems with this for a few months and have been slowly getting the fragments but i'm now down to a few that just cant seem to get since i get so few white colonies. also if it matters, some fragments i PCR'd gave more white colonies than others.

#2 Adrian K

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Posted 03 April 2010 - 12:20 AM

Dear molstudent I would like to help you but I do appreciate if you can be more clear in state your problem:

you mentioned:"they were very messed up at the ends"....what you mean by that? sequencing results?

For cloning efficiencies, sometimes it will be good and sometimes it is not. Thus you will get more blue at certain time and more white at the other. Also, check your TOPO competent cells. I'm afraid that your freezer is not cool enough and there might be defrost and refrost cycle which caused the TOPO vectors to be spoilt. Also, check the expiry date of your kits.

If you afraid your "A" ends is not good enough, try incubate 72C longer at the end of your PCR.

BTW, what is your fragment size?
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

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#3 molstudent

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Posted 03 April 2010 - 09:41 AM

Dear molstudent I would like to help you but I do appreciate if you can be more clear in state your problem:

you mentioned:"they were very messed up at the ends"....what you mean by that? sequencing results?

For cloning efficiencies, sometimes it will be good and sometimes it is not. Thus you will get more blue at certain time and more white at the other. Also, check your TOPO competent cells. I'm afraid that your freezer is not cool enough and there might be defrost and refrost cycle which caused the TOPO vectors to be spoilt. Also, check the expiry date of your kits.

If you afraid your "A" ends is not good enough, try incubate 72C longer at the end of your PCR.

BTW, what is your fragment size?


Sorry about not being clear, when i said the ends were messed up i did mean by sequencing results. all the expiration dates are fine and i even tried topo vector from another labs fridge that i know they had success with. also my control showed that the vector and cells are fine.

my fragment sizes ranged from 120bp to 1800bp and i'm down to a few that are 1100bp, 1700bp and 1800bp. I've already gotten some of the fragments of the same size though. i really dont know why these topo's arent working. the only thing i can think of is that the primers are messed up since the control from the topo kit worked. i also dont think its my templates because i always get a nice, clean looking band after my PCR. i got new primers in and will try those. i'll post back the results.

Edited by molstudent, 03 April 2010 - 09:42 AM.


#4 Adrian K

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Posted 04 April 2010 - 10:45 PM

LOL... now i understood.

for sequencing PCR products >1k bp, is a common thing that you got the end sequences being messed up. You will only get first few hundreds correct. Thus you need forward and reverse primer sequencing and build alignment with it (with one being reversed-complement). This is not with the problem of your TOPO.

Also, you can ask the sequencing company to do gene walking in order for you to get the full fragment of your PCR product. Re-order of primers is not necessary in this case, because it will not change much of the situation. Is the problem with the sequencer.

Hope this clarify all.
Adrian
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#5 molstudent

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Posted 05 April 2010 - 06:34 AM

Sorry if i have been unclear again, but i do have both forward and reverse primers and a lot of the "messed up ends" is the beginning of the fragment. the sequencing primers wouldn't explain my very very low topo hit rate of white colonies either. ill see how the new primers for PCR worked in a day or two.

#6 molstudent

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Posted 09 April 2010 - 12:11 PM

I tried the new primers and still same result. Only getting 1 or 2 white colonies per plate and they are negatives. I cant figure out why my PCR product isn't getting into the TOPO vector. Since i run it on a gel and purify it i know my fragment is the correct size so the PCR worked. what is causing this?

#7 rkay447

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Posted 09 April 2010 - 01:47 PM

I tried the new primers and still same result. Only getting 1 or 2 white colonies per plate and they are negatives. I cant figure out why my PCR product isn't getting into the TOPO vector. Since i run it on a gel and purify it i know my fragment is the correct size so the PCR worked. what is causing this?

Have you run a little of your pcr product on a gel after purifying? Also, I've never had this problem but I've heard gel purifying may destroy the A overhang. Perhaps it is worth reincubating the pcr product with Taq after gel purifying and then doing a quick column cleanup to get rid of the enzyme.

Edited by rkay447, 09 April 2010 - 01:47 PM.


#8 phage434

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Posted 09 April 2010 - 02:28 PM

Your insert could be toxic, when present in correct form. You may be selecting for the incorrect insert.

#9 molstudent

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Posted 09 April 2010 - 03:21 PM

I tried the new primers and still same result. Only getting 1 or 2 white colonies per plate and they are negatives. I cant figure out why my PCR product isn't getting into the TOPO vector. Since i run it on a gel and purify it i know my fragment is the correct size so the PCR worked. what is causing this?

Have you run a little of your pcr product on a gel after purifying? Also, I've never had this problem but I've heard gel purifying may destroy the A overhang. Perhaps it is worth reincubating the pcr product with Taq after gel purifying and then doing a quick column cleanup to get rid of the enzyme.


When i did the control pcr that came with the topo kit (that yields a ~700bp fragment) i also gel purified it before topo cloning and it worked fine. I have run my pcr product after purification on a gel and they look fine.

#10 molstudent

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Posted 09 April 2010 - 03:24 PM

Your insert could be toxic, when present in correct form. You may be selecting for the incorrect insert.


The template I PCR'd from was a plasmid from bacteria and all I'm trying to do is to add on different restriction sites to the ends. Since this fragment was made in bacteria in the current vector its in, doesn't that mean it shouldn't be toxic?

#11 Adrian K

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Posted 11 April 2010 - 10:33 PM

I tried the new primers and still same result. Only getting 1 or 2 white colonies per plate and they are negatives. I cant figure out why my PCR product isn't getting into the TOPO vector. Since i run it on a gel and purify it i know my fragment is the correct size so the PCR worked. what is causing this?


Did this "1 or 2" colonies in your plate contain your "vector with pcr insert"?

Edited by adrian kohsf, 11 April 2010 - 10:33 PM.

Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#12 BryanC

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Posted 12 April 2010 - 05:53 AM

What is your insert:vector ratio in your ligation reaction? In some stubborn clonings, I have gone up to 6ul insert, and keeping the salt and vector the same as recommended.

Also, how long are you leaving the ligation at RT? Contrary to what the invitrogen manual says, I sometimes find that longer than 5 minutes is required to get my insert. I have even left a ligation at RT for up to 4 hours and had plenty of clones to work with. This same cloning that worked with 4 hour ligation failed twice with the recommended 5 minutes.

Edited by BryanC, 12 April 2010 - 06:32 AM.


#13 molstudent

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Posted 12 April 2010 - 01:19 PM

the 1 or 2 colonies sometimes have my insert and vector and sometimes they dont but more often they dont.

i am ligating for 30 minutes and im not sure of the ratio. i just use 1ul of salts and have tried .5ul and 1ul of vector and i fill the rest with pcr product (gel purified). i have also tried 1ul of vector and 1ul of insert and the rest water, with no salts. i get the same outcome for each method.




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