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Molecular Cloning Forum DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


problem with TOPO TA cloning....

problem with TOPO TA cloning.... - Molecular Cloning Forum

problem with TOPO TA cloning.... - DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


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  #11  
Old 11-10-2009, 08:17 PM
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Default Re: problem with TOPO TA cloning....

On the topic of TOPO cloning, the manual I have says to add .5 - 4 ul of PCR product and 1 ul of TOPO Vector (among other things). I was wondering if anyone knew whether there's a more precise ratio or an optimum ratio of vector:insert product that you can add, or if it doesn't matter at all.
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  #12  
Old 11-11-2009, 10:44 AM
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Default Re: problem with TOPO TA cloning....

Generally optimum mole ratios are supposed to be 3:1 for insert, but I have had no problems with as low as 0,5:1. Just be sure to prepare enough plates with transformants (6 or more) to get enough clones for example if you are doing a clone library since lower insert/vector ratios result in lower cloning yields. Additionally you can adjust this ratio by using less vector than specified in the protocol (thus also saving money). I am routinelly using 0,5 ul of TOPO TA vector instead of 1 ul and it works great.
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Old 01-10-2010, 11:13 PM
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Default Re: problem with TOPO TA cloning....

Does your polymerase give you the required TA-overhangs? and are you performing the
TOPO ligation directly after the purification of your insert?

sometimes I have incubated the TOPO-reaction longer time than the protocol
says with some improvement.

TOPO is really useful when you get it going...so good luck
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  #14  
Old 01-25-2010, 06:53 PM
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Default Re: problem with TOPO TA cloning....

This is unrelated to the topic, but for those of you doing TOPO cloning, our company's cloning design software is designed to work with TA and directional TOPO cloning.

Some of you may find the virtual cloning construct program useful.
Search for our web-demo on youtube (foolproof cloning design software) or google us--bioinfoman

Cheers
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  #15  
Old 01-25-2010, 07:10 PM
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Default Re: problem with TOPO TA cloning....

Have you checked the competent cells? Is your TOPO-TA kit working for other clonings? 1.2 kb is not a large insert and you really need very less PCR product for this kit. Platinum Taq generates the same 'A' overhangs are as Taq, so am not sure why you are doing another step with Taq. The prime culprit in these clonings if your competent cells going bad or vector not stored properly. Also, are you electroporating or doing a chemical Transformation?
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Old 01-28-2010, 04:54 PM
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Default Re: problem with TOPO TA cloning....

OK Here's the deal:


According to Invitrogen tech support, 1 uL of TOPO-TA vector contains 10 ng of DNA. Furthermore, the best molar ratio of PCR Product: TOPO vector is actually 1:1. I always do a calculation first and see how many ng of PCR product I need to use in the TOPO reaction, and I get almost entirely white colonies after blue/white selection.

Here's what I do:

Do PCR. If PCR is clean, just do PCR cleanup (Qiagen) to get rid of salts/dNTPs/primers, etc. If it's dirty I do gel extraction. I measure DNA concentration by A260 or run it out on a gel if it's too low.

Then before I do anything else I calculate: (10)*(kb PCR)/(3.9 kb TOPO) = __ng PCR product for reaction. Once I know that I scale up appropriately to a 10-25 reaction volume for adding 3'-A overhangs with Taq (I always use high fidelity polymerases for cloning). A simple 10 min @ 72C always does the trick.

If I need x ng PCR product for TOPO reaction, I plan my 3'A reaction to contain x ng/uL PCR product. This way, I add exactly 1 uL and it simplifies my life. I mean, you do whatever you want, but I highly recommend calculating how much PCR product you need to add to the TOPO reaction before doing it, it really helps out. I always transform into regular, cheap DH5alpha. No need for anything fancy (although Invitrogen does force the TOP10 on you).

Using too much PCR product in the TOPO kit is a bad idea. I've had to dilute PCR product 100-fold. It's not going to disappear. Less is definitely better than more. Too much PCR product will pretty much ensure you have all incorrect clones.

Last edited by clevermizo; 01-28-2010 at 04:57 PM.
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Old 04-25-2010, 08:58 AM
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Default Re: problem with TOPO TA cloning....

hey clevermizo ]..but i am quite confused about it..

what do you mean by scaling up to a 10-25 reaction volume for adding 3A overhangs with Taq.

also, what does it mean you say

"If I need x ng PCR product for TOPO reaction, I plan my 3'A reaction to contain x ng/uL PCR product."

how do you calculate that?
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  #18  
Old 05-11-2010, 05:46 PM
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Default Re: problem with TOPO TA cloning....

Well I usually use a calculator to help out . Let me review a typical cloning reaction.


SCENARIO I:

Suppose I want to clone a sequence of 1.5 kb into the TOPO vector (3.9 kb). I am using this for downstream applications, so I want to minimize any possible mutations by using a proof-reading polymerase (Phusion, Pfu, etc.).

So, I run PCR and I have a nice product, which I have purified (gel purification, etc.) and I measure its concentration. Let us suppose that my 1.5 kb purified PCR product has a concentration of 50 ng/uL.

Now, I need to add the 3'-adenine overhangs in order to perform TA cloning. To do this I use a standard Taq with 10 mM dATP (or 10 mM dNTP mix works fine) and 10X Taq Buffer.

I want to clone in my 1.5 kb product in a 1:1 molar ratio with the 3.9 kb TOPO vector. My TOPO vector is 10 ng/uL. So:

(10 ng TOPO) * (1.5 kb PCR product)
------------------------------------ = X ng PCR
(3.9 kb PCR product)

This means in my TOPO reaction, I will need to assemble:

10 ng TOPO + 3.8 ng PCR product.

So to simplify my life, I will plan my 3'-adenine Reaction to have a final concentration of 3.8 ng/uL. Thus my TOPO reaction will be:

3 uL H2O
1 uL Salt Solution (with TOPO kit)
1 uL 3.8 ng/uL Taq-treated PCR product
1 uL 10 ng/uL TOPO vector.
-----------------------------
6 uL total.


If I want a 25 uL Taq reaction with a final concentration of 3.8 ng/uL, then I need to seed it with 95 ng of purified PCR product. Since my original PCR product concentration above was 50 ng/uL, I will use 1.9 uL:

19.6 uL H2O
2.5 uL 10X Taq Buffer
0.5 uL 10 mM dATP (or 10 mM dNTP mix)
1.9 uL of 50 ng/uL Purified PCR product
0.5 uL Taq

I mix this up, spin it down and incubate it at 72C for 10 minutes. Then I assemble my TOPO reaction as above, using 1 uL of Taq-treated PCR product.

The whole point is so that the molar ratio of TOPO vector to Taq-treated PCR product is 1:1. This produces the most efficient ligations and works for me every single time, with almost all white colonies after transformation.


SCENARIO II:

If you do not need to use this for downstream cloning and you have been using Taq polymerase all along, then the addition of 3'Adenine is unnecessary.

Suppose then that I have 50 ng/uL of purified 1.5 kb Taq-amplified PCR product. Well, my optimal ratio is:

10 ng (1 uL) TOPO : 3.8 ng PCR product.

Now, I won't be able to pipet 0.076 uL, so I dilute my PCR product down to 2.5 ng/uL (20X dilution) in water, or Tris buffer or something. Then I can use ~1.5 uL of 20X diluted PCR product in my TOPO reaction:

2.5 uL H2O
1.0 uL Salt solution
1.5 uL 20X diluted purified Taq-amplified PCR product
1.0 uL 10 ng/uL TOPO vector
-----------------------------
6 uL total (5 minutes incubation @RT).

Last edited by clevermizo; 05-15-2010 at 09:50 PM.
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  #19  
Old 05-13-2010, 01:55 PM
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Default Re: problem with TOPO TA cloning....

what if my cloning sequence is 4.2kb long? and my purified PCR is usually >100ng/ul
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  #20  
Old 05-13-2010, 02:23 PM
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Default Re: problem with TOPO TA cloning....

oh never mind i think i got it...it's just really hard to transform in a sequence that big..
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