Failed TOPO cloning - what happened? - (Apr/03/2014 )

My PCR-product had been generated by Phusion. So I had to create the required 3' A-overhangs needed for the TOPO.

 

So I did the following:

- 10ul DreamTaq buffer

- 1ul 10mM dATP

- 5ul of the PCR-product (electrophoresis validated it and I had purified it previously to get rid of Phusion, primers etc)

- 0.2ul ( = 1unit) of DreamTaq Polymerase (5U/ul)

 

Incubated the solution at 72C, for 8-10min. No cycle/shaking thermo-mixer.

 

Placed sample immediately on ice and did the TOPO:

- 1ul of above sample (now with hopefully 3' A-overhangs)

- 1ul Salt Solution

- Water up to 5ul

- 1ul TOPO vector (pCR4 TOPO)

 

Kit is from Invitrogen

 

Mix gently

Incubate for 5min at 22C

Place on ice and do the transformation with DH5 competent cells

Plates 50ul on one plate, and remaining volume on another plate

 

Incubated at 37C but I got no growth the day after. Nothing!

 

Any inputs?

Also, what is the maximum volume that can be used when plating competent bacteria on agar selection-plates?

I took 50ul on on plate which was a good volume, but the remaining volume is close to 300ul. And thus the plates get really really wet so to say.

 

How did your controls look? 

Since the PCR had already been done previously, I had no PCR control.

For the TOPO cloning, there were no growth on plates either.

 

Does the incubation-time, temp and used volumes look reasonable?

The protocol stated incubate for 5min at 22 - 23C. So I put the sample in a thermo-shaker/cycler at 22C (no shaking). Should I instead have incubated the sample at regular room-temperature?

The kit is from Invitrogen. pCR4-TOPO and DH5 competent cells.

 

Can someone explain why TOPO cloning protocols just state volumes to be used, and not CONCENTRATIONS in the mixture?

So the Control TOPO cloning reaction (from the kit) or/and Transformation Control (from the kit) also grew no colonies or you didn't have any?
 

To TOPO TA cloning is usually piece of cake with a standard length non problematic PCR product (300-500bp), so 5 minutes at RT or 22 or half an hour or whatever makes no difference if you got zero colonies.

Without controls you can't say where the problem was.

I had no controls at all.

 

I am gonna do it fresh tomorrow. And this time use DreamTaq for the PCR so that I get the 3' T-overhangs in the PCR.

 

Question 1) Which approach is the best for purification: a) or b)

a) run a small sample on electrophoresis (5ul). If I get a clear band I will then purify the other PCR-sample (45ul) that I did not run on agaros electrophoresis

b) run 2 electrophoresis, one with 5ul and one with 45ul. The one with 5ul I expose to UV to see whether I get a clear band, and if I do then I purify the PCR-product from the other agars-electrophoresis (which I save from UV-exposure).

 

I am thinking that a) is a more preferable approach than b) since purification from a solution would give BETTER YIELD than purification from agaros?

Any comments on this?

 

Question 2)

Also, I could use less than 45ul for the purification, maybe 20ul or 30ul and save the remaining volume in the -20C for future use if necessary, so that I don't have to do another new expensive PCR. Sounds like a good plan? Or just use everything?

 

Question 3)

The PCR-reaction is with Taq-polymerase in mind.

But I am gonna go with DreamTaq and therefore need to set my PCR-cycle settings according to DreamTaq. The temperature and times will be different. Will that cause a problem?

 

Question 4)

The Taq-PCR protocol for the TOPO states that only use between 10-100ng DNA for the PCR.

But the DreamTaq manufacture sheet recommends to use between 0.01ng - 1ng. Which is a huge difference from 10-100ng.

If I follow the DreamTaq guidelines and only use 1ng for the PCR, would that screw up the TOPO later. Be too little DNA-insert?

 

Of if I use 1ng for the PCR, and the electrophoresis shows a strong clear clean band, then I can rest assure that 1ul of this purified PCR-product will work just fine for the TOPO?

 

My other concern with this is that 1ng might be as little volume as 0.25ul. Which can be hard to pipett.

1) Yes, I wouldn't think gel extraction is necessary for this, if you didn't needed overhangs you would just use plain PCR reaction without any purification in ligation.

 

 

2) Since the original protocol uses just volume of PCR reaction, you should not exceed recommended volume (may be important for the ligation solution) but try to use roughly equivalent amount of DNA as you would have by just using PCR reaction right away (purification recovers around 80 % of DNA, but often elutes in different volumes than original PCR was).

 

 

3) You use Phusion for PCR, right? Then use the Phusion settings to get efficient amplification, first. Then, for the overhangs use just the 72 degs, UNLESS DreamTaq is some kind of hotstart enzyme, then it woudn't be active without denaturation step.

 

 

4) 10-100 ng is standard PCR template starting amount, since you amplify with Phusion, look for Phusion recommendations. DreamTaq only makes the overhangs, it doesn't amplify anything, its starting template amount is hardly relevant. But if the overhangs still wouldn't work, then consider getting somewhere a bit of stupid simple old Taq and see if changes anything. Who knows what they put into these boosted Taqs nowadays.. you just want overhangs..

 

 

 

 

(I have a protocol to make A overhangs just by adding 1 U of Taq for 15 minutes on 72 deg at end of the PCR. It should work in the Phusion buffer and dNTPs are there anyway. Phusion will try to correct overhangs, but theoretically Phusion will be a bit exhausted after the PCR, and probably 5 min at 95 deg before adding Taq, will kill the most of what's left. The fresh Taq should make enough overhangs then. But I personaly didn't try this protocol.)

I'm with Trof. Do your normal PCR (if necessary verify you have a clean single band). Add 1 ul Taq directly to your PCR product. You might also want to add a bit of dATP (not ATP). Incubate at 72 for 5 minutes, then do the Topo reaction. There is also a Topo-blunt kit (never tried it) which works with blunt fragments.

We had pCR4Blunt-TOPO vector for blunt cloning. It's great that it kills bacteria transformed with plasmid without your insert, since it interupts ccdB gene in the vector (unsuitable for very short inserts or those that would leave the lethal gene functional though).

 

Unfortunatelly for me then, I used a Taq-derived enzyme and had to digest the overhangs ;)

I have tried for two months all the available protocols for adding the ovehand A's after a PCR with a Pfu enzyme and it didn't work. Don't bother to try; it is much much easier to ask for a mix of Taq and a proofreading enzyme than spending vials and vials of competent cells. Good luck.

@ Adonyire - its worked for me every single time I have tried it.