5 replies to this topic

[particle adventurer]

Hi,

I have been attempting TOPO cloning with the invitrogen TOPO-TA Cloning kit.
However a senior was rather daunting and bullying refuse to let me proceed when I once tried to use an overnight stored PCR product for Ligation reaction and transformation.

Is it absolutely necessary to have a fresh PCR product for TOPO cloning?
What happens if I use one that is stored overnight?

How stable are the A overhangs incorporated by Taq Polymerase in PCR coz that is the basic principle which TOPO cloning exploits?

Also is electroporation better or chemical transformation better for TOPO clones?

Finally, which is the better method to purify PCR products: Gel elution or PEG purification and why? Coz he absolutely insists its gel elution whereas i use PEG

I know it would take some effort to reply to each of these questions. However plz lend a hand to this fellow labbie to steer through the molecular maze ...

Cheers,
Particle Adventurer

[bob1]

Overnight is the very maximum you would want to use.  I suggest using fresh, it works a lot better than stored PCR product.

[Adrian K]

particle adventurer, on 23 August 2010 - 07:44 AM, said:

Hi,

I have been attempting TOPO cloning with the invitrogen TOPO-TA Cloning kit.
However a senior was rather daunting and bullying refuse to let me proceed when I once tried to use an overnight stored PCR product for Ligation reaction and transformation.

Is it absolutely necessary to have a fresh PCR product for TOPO cloning?
What happens if I use one that is stored overnight?

How stable are the A overhangs incorporated by Taq Polymerase in PCR coz that is the basic principle which TOPO cloning exploits?

Also is electroporation better or chemical transformation better for TOPO clones?

Finally, which is the better method to purify PCR products: Gel elution or PEG purification and why? Coz he absolutely insists its gel elution whereas i use PEG

I know it would take some effort to reply to each of these questions. However plz lend a hand to this fellow labbie to steer through the molecular maze ...

Cheers,
Particle Adventurer

First of all, your senior done the right thing.
If I'm your senior I would do the same because he/she don't want you to waste.
Although is not necessary, but "A" overhang is not stable for a stored overnight PCR products. You will get very less ligation efficiency.
I can't comment on Electroporation as I never done before, but I do understand TOPO does provides the competent cells, and all you need is just add in and done.
Gel-elution/gel-excise purification is always the best method, but bear in mind you shouldn't expose your band too long under UV (which will loosen-up the "A" overhang or destroy the DNA). The reason for doing gel-excise is because you want to get rid of unspecific band (some which is unseen). Your senior should be able to guide you on how to do a gel-excise under minimum UV exposure, although he/she might be mean, but your senior don't want you to waste, and your senior want you to learn the right thing.

Cheer up as you got a good senior as your mentor.

Adrian

Edited by adrian kohsf, 23 August 2010 - 08:14 PM.

[sera_tonin]

i recently did a TOPO reaction: i ran my PCR product on a gel, cut out my bands, and stored them at 4-C for two nights (this was saturday). monday, i did a gel recovery followed by the TOPO reaction and electroporation (i've never done transformation with TOPO, so i don't know how well that works). i didn't get tons of colonies, but about 80% of the colonies i got had my insert, in the correct orientation. maybe doing it with fresh product would have increased the efficiency, but i didn't have any major problem, not using fresh product. but in my experience, it's best to just do what your superiors tell you, even if there's another way - some people are stuck in their ways and refuse to hear that other options work!

[Tina Hansen]

I've used old PCR product before. If it is really old (>month), I usually add som Taq and elongate for 10 min 72C to re-establish the A-overhangs before cloning. It has worked fine.

[OA17]

particle adventurer, on 23 August 2010 - 07:44 AM, said:

Hi,

I have been attempting TOPO cloning with the invitrogen TOPO-TA Cloning kit.
However a senior was rather daunting and bullying refuse to let me proceed when I once tried to use an overnight stored PCR product for Ligation reaction and transformation.

Is it absolutely necessary to have a fresh PCR product for TOPO cloning?
What happens if I use one that is stored overnight?

How stable are the A overhangs incorporated by Taq Polymerase in PCR coz that is the basic principle which TOPO cloning exploits?

Also is electroporation better or chemical transformation better for TOPO clones?

Finally, which is the better method to purify PCR products: Gel elution or PEG purification and why? Coz he absolutely insists its gel elution whereas i use PEG

I know it would take some effort to reply to each of these questions. However plz lend a hand to this fellow labbie to steer through the molecular maze ...

Cheers,
Particle Adventurer

Hi! From my experience, the procedure depends very much on the yield you expect: if you want to use topo for cloning a library, you will need a very high efficiency in all the steps (PCR, cloning and transformation), and you will need to be very careful. But if you only need one colony, you don't have to optimize the steps to such an extent. Here are my answers to your questions:

1) It is not absolutely necessary to have a fresh PCR product in order to clone into the Topo vector. However, you will get better results if the PCR product is fresh. The last time I did a Topo cloning, I used a PCR product that had been purified with a column kit, and it had been stored at -20ºC for several days, and it worked pretty well!! However, if you need a high cloning efficiency, I would always use a fresh product. If you just need one good colony, then go ahead and don't worry.

2)I think that the A overhangs are not extremely stable, but if you do not need a high cloning efficiency, freezing or purifying the PCR product will not do so much damage.

3)In general, electroporation gives a higher yield of transformants because electrocompetent cells usually have a higher efficiency than the others. Since the topo vector is small, it should be easily transformed into any kind of cells, as long as the efficiency is good enough (not less than 10E7 ufc/µg). I have done chemical transformation with good results.

4)Depends on the band that you have got from your PCR: if it is bright and sharp, or if you have subproducts (that sometimes you can't even see!). For Topo, I know some people who don't even purify the product: they clone straight away after the PCR reaction has finished.

Good luck!!