Cannot clone 2kb insert into pENTR D TOPO cloning vector - (Oct/24/2013 )
Hello everyone,
My first post on protocol-online.
I've got a problem with Invitrogen's pENTR D TOPO cloning kit. My problem = I can't seem to get a 2kb PCR amplicon cloned into the kit's entry vector (this is supposed to occur during the 5 minute "TOPO Cloning Reaction").
Theprocess
A) I've got a PCR'ed up amplicon which I want to insert into the entry vector (verified size of amplicon on agarose gel electrophoresis)
B> Invitrogen D-TOPO kit advises a 5 minute 'TOPO Cloning Reaction' which I perform.
C) I then follow the instructions and transform the entry clone into chemically competent E. coli which are then plated onto kanamycin agar plates
D) Colonies are then chosen, cultured, and then the plasmid DNA is purified and sequenced.
The sequencing data reveals that the E. Coli were transformed with the D-TOPO plasmid not containing the PCR amplicon. This suggests that Step B (TOPO Cloning Reaction) has failed. I have done this twice already.
For the TOPO Cloning Reaction, the instructions advises use of 0.5uL to 4uL of PCR product containing 5-10ng of DNA (for 2kb product). I have so far used 10 and 20ng of DNA with no success. Should I attempt higher amounts?
Any advice would be greatly appreciated! Thanks
Are you using a Taq based PCR enzyme? High performance enzymes such as Phusion or Q5 will not work in this reaction.
Hi Phage434
Thanks for the comment.
phage434 on Thu Oct 24 12:25:59 2013 said:
Are you using a Taq based PCR enzyme? High performance enzymes such as Phusion or Q5 will not work in this reaction.
I used Phusion as the invitrogen pENTR-DTOPO manual (website here; pdf here) specifically states that one can use any thermostable, proofreading polymerase for the PCR process.
I did not use Taq as it lacks proofreading function (the manual says one should not use Taq).
Surely if what you are saying is true, it would be highlighted in the manual?
I'm sorry, I stand corrected. Your kit is using the blunt cloning version of the TOPO vectors, which allow cloning of blunt ended PCR products. The standard TOPO TA vectors required the addition of the A base produced by Taq polymerase. The Phusion you are using should work. Are you adding salt to the TOPO reaction?
pepi on Thu Oct 24 12:11:39 2013 said:
The sequencing data reveals that the E. Coli were transformed with the D-TOPO plasmid containing the PCR amplicon.
Isn't this what you want?
Hi phage434, thanks again for the post.
Indeed I am using the salt. Here is the recipe for the 'TOPO cloning reaction' as per the manual (linked in my first post)
A) PCR product 0.5-4 uL
B> Salt solution 1uL (fixed volume)
C) Topo cloning vector 1uL (fixed volume)
D) sterile water to 6uL total
The manual reads "For optimal results, be sure to use a 0.5:1–2:1 molar ratio of PCR product:TOPO® vector in your TOPO® Cloning reaction." It also reads that use of "5–10 ng of a 2-kb PCR product in a TOPO® Cloning reaction generally results in a suitable number of colonies."
The problem with the above is that I have no indication regarding the actual concentration/amount of enzyme in the TOPO vector, hence it becomes impossible (?) to determine the molar ratio. Or am I missing something obvious?
I have tried using a PCR product containing 10ng as well as 20ng of DNA. With both, the 2kb insert does not enter the entry vector and I end of transforming E coli with empty plasmids.
HomeBrew on Thu Oct 24 20:21:22 2013 said:
pepi on Thu Oct 24 12:11:39 2013 said:
The sequencing data reveals that the E. Coli were transformed with the D-TOPO plasmid containing the PCR amplicon.
Isn't this what you want?
Sorry HomeBrew, that is a mistake in my post. I have now corrected it. It should read "The sequencing data reveals that the E. Coli were transformed with the D-TOPO plasmid NOT containing the PCR amplicon."
The ratio refers to the amount of vector to the amount of insert, i.e. 1:1, 1:3, etc...
You have used this kit with the provided control and are sure that it works?
I have used sticky end based TOPO kits and have had varying success depending on the gene. Double check and make sure you are correctly calculating the concentration/ratio of your vector and insert. If varying the ratio of the vector:insert doesn't work, I would consider increasing the TOPO reaction time.
You could feed your insert though a secondary structure prediction program and see if it is forming a weird structure that is impeding proper ligation... but this is probably has nothing to do with your problem.
How many colonies did you test?
Hi Jerryshelly1, thanks for the comments and advice.
1) I have not used the provided control. This is something I should do.
2) I will re-calculate the amount of PCR product I have
- I did the above by comparing the densitometry of the PCR channel to the DNA marker channel on the agarose electrophoretic gel. (The amount of DNA in the bands in the marker are known)
- Is there a better way of doing this? (I PCR'ed the amplicon from a plasmid)
3) I will increase the TOPO reaction time, perhaps that is the problem?
4) Regarding the colonies: I had very poor growth - only 3 colonies in total during my first run and 4 colonies on the subsequent run (plated on kanamycin-agar plates). This makes me suspect that both the TOPO cloning and the transformation were problematic.
You could nanodrop your PCR sample or use a DNA specific fluorometer for an exact reading; however, using HindIII for densitometery is an alright method.
Increasing the reaction time could potentially help you. You may just run the reaction until the enzymes are dead, but it shouldn't hurt you overall.
Sometimes colleagues prepare the wrong concentration of antibiotics. Double check and make sure you are adding the appropriate concentration.
Can you share the gene you are trying to TOPO?
Edit - One more question... Can you cut out your gene from your other plasmid using a blunt RE? Phusion polymerase is amazing, but maybe something funky is going on.