Directional TOPO Cloning

[kdawg]

Hi,
I've been trying for months now to produce 3 separate clones using the pENTR/D-TOPO directional cloning kit from invitrogen with absolutely no success. I would any and all input on the subject. Here we go
1. PCR using Pfu polymerase fragment (1kb - 1.5kb) using double checked primers with cacc- immediately before the ATG
2. gel extract and purify (promega wizard kit)
3. have followed this with ethanol precipitation
4. measured A260 and calculated DNA concentration of PCR product to calculate 1:1 molar ration vector to insert
5. performed TOPO cloning protocol and transformation (using both Mach1 and Top10 cells - very gently)
6. here's the tricky party - get HUNDREDS of colonies
7. Culture, miniprep and digest with Not1 (which doesn't cut any of my inserts) (used a cut empty vector as comparison)
8. - NO INSERT!!!! all bands are the same size as the cut entry vector (although the last one I did had all bands at 1.5kb and the cut empty vector at 2580kb - go figure)

about to attempt pCR8/GW/TOPO upon receipt of new primers but this is really doing my head in.
I have my heart set on the TOPO system for ease of use of downstream projects i.e. expression of gene in mammalian and bacterial cells, protein purification, site-directed mutagenesis etc. so I REALLY want this to work.

If anyone had any hints or tips I'd be most appreciative!

[abilei]

Hi Kdawg, I read your post about your problems with directional topo cloning and found out that it's exactly the same problem I have!!
Did you eventually find a solution? Any trick that could help me a little bit? ...because at this point I'm running out of ideas!!!!

[kdawg]

i did eventually get my clone - i just tried tweaking with the protocol a bit - doin half rxns etc (to save vector). but it the end it took colony PCR'ing i think 48 clones and i found 2 positives, grew them up, sequenced them and thankfully they were correct. i'd advise if you're doing everything by the book just colony PCR around 30 colonies and if you get a band grow them up and check them via digest and sequncing. hope this helps, but no golden trick because i've used this same system at the same time with other genes and haven't had a single problem. some genes are just annoying.
one thing though - have you checked your kit? because one of my kits were faulty and invitrogen replaced it free of charge - just do the control reactions.

[Helen Troilo]

You could probably save yourself a LOT of time looking up an alkaline phosphatase protocol to avoid self-ligation. There might be a few colonies amoung the hundreds with your insert in but its unlikely you'll find them with self-ligating vector. Also 3:1 insert to vector ratio is more usual than 1:1.

[abilei]

Originally Posted by kdawg

i did eventually get my clone - i just tried tweaking with the protocol a bit - doin half rxns etc (to save vector). but it the end it took colony PCR'ing i think 48 clones and i found 2 positives, grew them up, sequenced them and thankfully they were correct. i'd advise if you're doing everything by the book just colony PCR around 30 colonies and if you get a band grow them up and check them via digest and sequncing. hope this helps, but no golden trick because i've used this same system at the same time with other genes and haven't had a single problem. some genes are just annoying.
one thing though - have you checked your kit? because one of my kits were faulty and invitrogen replaced it free of charge - just do the control reactions.

The kit is ok, I performed the control reactions and everything was fine. I cloned it in two different conditions and got 7 out of 10 and 3 out of 10 positive and right clones.
Nothing to do with my gene. I picked 30 colonies with no success. As you say some genes are just annoying...
I'll try to save some vector doing half reactions as you say.
Can you tell me how do you do the colony PCR? because I've tried a couple of times but it didn't work. Maybe too much DNA? How much of the colony do you use? Can you pass me your protocol?
Thanks a lot!

[abilei]

Originally Posted by kdawg

i did eventually get my clone - i just tried tweaking with the protocol a bit - doin half rxns etc (to save vector). but it the end it took colony PCR'ing i think 48 clones and i found 2 positives, grew them up, sequenced them and thankfully they were correct. i'd advise if you're doing everything by the book just colony PCR around 30 colonies and if you get a band grow them up and check them via digest and sequncing. hope this helps, but no golden trick because i've used this same system at the same time with other genes and haven't had a single problem. some genes are just annoying.
one thing though - have you checked your kit? because one of my kits were faulty and invitrogen replaced it free of charge - just do the control reactions.

Thanks for you reply kdawg. I'll try with half reactions to save some vector, but i do think that some genes are a pain to be cloned as you said.
Could you pass me the protocol for colony PCR? I found hundreds of them in the web and tried a couple of them with no success. I have the feeling that the DNA amount is the problem...

[primer3]

Hi everyone, does any of you having encountered a problem like this: everything was right, from PCR to insert vector ratio, just can not get a single colony? I have spent one months doing topo cloning, check back all the possible factors again and again, but just can not get a clonoy, who can help me out!