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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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  #21  
Old 05-13-2010, 07:48 PM
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Default Re: problem with TOPO TA cloning....

Quote:
Originally Posted by hhhsandy View Post
what if my cloning sequence is 4.2kb long? and my purified PCR is usually >100ng/ul

Well, then use the appropriate 1:1 molar ratio with the 3.9 kb TOPO vector and it should work. I've cloned in large things before.

The molar ratio is key in any ligation. Do you want me to do the calculations for you or something? Because, if you just use your numbers in my scenarios above, you should be fine.
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  #22  
Old 05-14-2010, 02:29 PM
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Default Re: problem with TOPO TA cloning....

oh..i think i got the numbers..just that i was wondering if I could just use 5ul of taq instead of 25ul..cuz afterall, if you are just taking 1ul from that mix to make the TOPO..wouldnt it be kind of a waste to make such a big mix?
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  #23  
Old 05-14-2010, 04:55 PM
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Default Re: problem with TOPO TA cloning....

Quote:
Originally Posted by hhhsandy View Post
oh..i think i got the numbers..just that i was wondering if I could just use 5ul of taq instead of 25ul..cuz afterall, if you are just taking 1ul from that mix to make the TOPO..wouldnt it be kind of a waste to make such a big mix?

Sure, you can scale that down if you want. And I think you mean the Taq reaction. No one's using 25 uL of Taq polymerase (What is that, like 250 Units ?) I just use 25 because it's convenient. Can you really pipette 0.1 uL of taq polymerase for a 5 uL reaction accurately? Or how about 0.1 uL of dATP? Because I know I sure can't. Even with the P2. You can't really even see those volumes. You could dilute all the reagents first I suppose or make a bigger mix and aliquot it, but then that would defeat the purpose of your desire to save reagents.

Personally I try never to pipette less than 0.5 uL of anything.

Remember you don't need to add the 3'A overhangs if you did your original PCR with a Taq polymerase. That's only necessary if you did the original PCR with an error-correcting polymerase like Pfu. If you used a Taq polymerase in the first place, the 3'A overhangs are already there. Just put the right ng amount of purified product in your TOPO reaction and adjust the water accordingly.

Last edited by clevermizo; 05-14-2010 at 05:03 PM.
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  #24  
Old 05-15-2010, 06:56 AM
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Default Re: problem with TOPO TA cloning....

Quote:
Originally Posted by clevermizo View Post
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:

9.6 uL H2O
12.5 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).
ok..just to make sure..

why are you using 12.5 ul of 10X buffer?
shouldnt it be 2.5ul since it's 10x? and you want your total volume to be 25ul?
and 0.5ul for taq?

btw, i didnt use taq in my PCR reaction, so I am sure i need this step!

Sorry, for having so many questions! >.<

but i really do need your explanations!
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  #25  
Old 05-15-2010, 09:49 PM
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Default Re: problem with TOPO TA cloning....

Quote:
Originally Posted by hhhsandy View Post
ok..just to make sure..

why are you using 12.5 ul of 10X buffer?
Oops! That was a typo. It should be 2.5. Sorry about that. I'll edit my previous post.

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

Now remember, the "1.9 uL" was dependent on my example. In general it should be : (25 * X ng)/Y(ng/uL) where X is the amount of ng you determined will be 1:1 molar ratio with 10 ng of TA vector, and Y is the PCR product concentration. 25 is just "Taq treatment" reaction volume, so that the "Taq Treatment" reaction will be X ng/uL final concentration. If you are scaling that down, then for example (10*X)/Y for a 10 uL reaction.

Another example. Suppose I need to use 3 ng of a 40 ng/uL PCR product in ligation with 10 ng of TA vector. Well:

X = 3ng
Y = 40 ng/uL

And to save reagents, I want to use a 10 uL Taq Treatment Reaction volume. So, I need to use: 10*3/40 = 0.75 uL. See? This is what I meant about pipetting.

Anyway, going with this then:

7.85 uL H2O
1.0 uL 10X Taq buffer
0.2 uL dATP (10 mM)
0.2 uL Taq (mine is 5U/uL)
0.75 uL PCR product

This will be 3 ng/uL final concentration, and I can simply use 1 uL in the TOPO reaction.

And you are correct. If you did not use a Taq polymerase to perform PCR, you cannot do TA cloning until you treat the sample with Taq polymerase this way. (That's true for any TA vector, not just TOPO. pGEM-T vectors are another set of TA vectors. The TOPO vectors simply make the ligation more efficient by having the TOPO isomerase enzyme linked to the vector).

Last edited by clevermizo; 05-15-2010 at 10:00 PM.
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  #26  
Old 05-17-2010, 03:15 PM
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Default Re: problem with TOPO TA cloning....

Quote:
Originally Posted by clevermizo View Post
Oops! That was a typo. It should be 2.5. Sorry about that. I'll edit my previous post.

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

Now remember, the "1.9 uL" was dependent on my example. In general it should be : (25 * X ng)/Y(ng/uL) where X is the amount of ng you determined will be 1:1 molar ratio with 10 ng of TA vector, and Y is the PCR product concentration. 25 is just "Taq treatment" reaction volume, so that the "Taq Treatment" reaction will be X ng/uL final concentration. If you are scaling that down, then for example (10*X)/Y for a 10 uL reaction.

Another example. Suppose I need to use 3 ng of a 40 ng/uL PCR product in ligation with 10 ng of TA vector. Well:

X = 3ng
Y = 40 ng/uL

And to save reagents, I want to use a 10 uL Taq Treatment Reaction volume. So, I need to use: 10*3/40 = 0.75 uL. See? This is what I meant about pipetting.

Anyway, going with this then:

7.85 uL H2O
1.0 uL 10X Taq buffer
0.2 uL dATP (10 mM)
0.2 uL Taq (mine is 5U/uL)
0.75 uL PCR product

This will be 3 ng/uL final concentration, and I can simply use 1 uL in the TOPO reaction.

And you are correct. If you did not use a Taq polymerase to perform PCR, you cannot do TA cloning until you treat the sample with Taq polymerase this way. (That's true for any TA vector, not just TOPO. pGEM-T vectors are another set of TA vectors. The TOPO vectors simply make the ligation more efficient by having the TOPO isomerase enzyme linked to the vector).
ok! thanks! cant wait to try the new construct! i will let you know the result
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  #27  
Old 05-19-2010, 01:57 AM
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Default Re: problem with TOPO TA cloning....

i am still not getting many colonies...maybe 2? arrgg!!
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  #28  
Old 05-19-2010, 05:24 PM
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Default Re: problem with TOPO TA cloning....

Quote:
Originally Posted by hhhsandy View Post
i am still not getting many colonies...maybe 2? arrgg!!
How much of the TA reaction do you transform? How much do you plate? What's your transformation procedure?
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  #29  
Old 05-20-2010, 03:18 PM
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Default Re: problem with TOPO TA cloning....

I PM you..just dont want to spam this place with my msg! :P
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  #30  
Old 05-21-2010, 01:49 PM
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Default Re: problem with TOPO TA cloning....

Do you know what spam is? If you have a question or a message that is on-topic it may simply help other people who have the same problem.

Spam is advertising.
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