15 replies to this topic

[crackanaut]

Hello all, I'm quite new at cloning.. i'm using a kit from invitrogen. the pcr product i want to clone is 5 kb and i'm using chemically competent cells which i've stored in a -80 freezer.  My first two trials yielded no results-no colonies- so i began to doubt the competency of the cells (there have been power failures), the second time around i plated some on Macconkey. I got a few colonies and I've inoculated those into LB, hoping its the strain I want. Can someone suggest a simple do-it-urself method for transforming this culture? Can I try electroporation with the same? Also, I've more vials of competent cells left in the freezer but i suspect that there are only a few viable competent cells in the vials of the kit-would it be better if I incubated the vector-cell mixture in SOC medium after transformation, for a day before plating out on LB kanamycin plates?  Many thanks..

Edited by crackanaut, 17 March 2009 - 06:35 PM.

[noelmathur]

Honestly, I am not very clear how are you trying to clone your insert, is it TA cloning? or which other kit are you using? Without knowing what are you using, I doubt anybody can help you.

Edited by noelmathur, 17 March 2009 - 11:12 PM.

[almost a doctor]

What makes you think is the transformation that is failing, and not any of the other steps?  

I agree with noelmathur, we need more information to be able to help you, like a detailed protocol of what you are doing.

[crackanaut]

i'm trying TA cloning; according to the protocol- you only need to mix the vector and the purified product for 5 minutes, and claims that you can then mix this with the vial of competent cells. Rest of the protocol involves incubation on ice for 30 min, heat shock, and addition of SOC medium. The cells are incubated in a shaker for an hour and then plated on to LB kanamycin. The reason i suspected competent cells is because i've followed all instructions to the letter and its too simple and straightforward for a major goof up.

[Clare]

crackanaut, on Mar 18 2009, 02:56 PM, said:

i'm trying TA cloning; according to the protocol- you only need to mix the vector and the purified product for 5 minutes, and claims that you can then mix this with the vial of competent cells. Rest of the protocol involves incubation on ice for 30 min, heat shock, and addition of SOC medium. The cells are incubated in a shaker for an hour and then plated on to LB kanamycin. The reason i suspected competent cells is because i've followed all instructions to the letter and its too simple and straightforward for a major goof up.

There's still a few things that you have not mentioned that could go wrong...

Does your polymerase generate overhangs? Some don't   Also, are you setting up the reaction ASAP after the PCR?
Clare

[perneseblue]

crackanaut, on Mar 18 2009, 06:56 AM, said:

The reason i suspected competent cells is because i've followed all instructions to the letter and its too simple and straightforward for a major goof up.

You will be surprised.

Can you tell us how was the PCR fragment made? Specifically what polymerase did you use? Was it a polymerase mixture that contain Taq? Or did you use a proof reading polymerase then treat it with Taq?

TA cloning requires the DNA fragment to contain an A overhang.

As for your question, no you can not use chemical competent cells in an electroporation. The chemical competent cells are treated differently and carry too much salt for electroporation.

And no, you should not recover your cells in SOC for longer than 1hr before plating them on selection. Without selection, the untransformed cells will have a huge growth advantage and will dominate the culture.

[jiajia1987]

I am sorry to add on to this.

But I would like to know if anybody has encountered the same problem as me.

I did a PCR with Taq polymerase and did a TA cloning. Before the TA cloning, I did PCR cleanup. After the transformation, I picked some colonies and did sequencing. Other than my inserts, I got many other inserts which I didnt want at all. And most of them were less than 100bp. My gene is 1.5kb and I have heard that inserts smaller than 100bp ligate better in TA cloning.

Has anyone encountered this?

[crackanaut]

I did use a predominantly Taq based enzyme- the kit mentioned that any enzyme system which has a higher proportion of Taq is compatible with theirs and I verified with the enzyme providers too and they said its ok with TA cloning. I managed to get just a few colonies when i plated the competent cells directly on to MacConkey agar, I have pinned my hopes on reviving a culture and then getting it transformed with CaCl? What wud be my odds for that?

As for the time gap b/w pcr and cloning, the first time around I cleaned up the PCR product and stored it at -20 for a few days. The second time, I proceeded directly to cloning after eluting the product from agarose

Edited by crackanaut, 18 March 2009 - 11:19 PM.

[noelmathur]

jiajia1987, on Mar 19 2009, 03:35 AM, said:

I did a PCR with Taq polymerase and did a TA cloning. Before the TA cloning, I did PCR cleanup. After the transformation, I picked some colonies and did sequencing. Other than my inserts, I got many other inserts which I didnt want at all. And most of them were less than 100bp. My gene is 1.5kb and I have heard that inserts smaller than 100bp ligate better in TA cloning.

Has anyone encountered this?

I am not too sure if your PCR is working out well. Try running PCR product on 2% agarose gel and see your band of interest plus others, gel purify your band of interest, add A overhang by incubating with Taq and salts at 70°C for 10 mins and then setup TA-cloning.

I am not great fan of TA cloning due to their less efficiency.

[noelmathur]

crackanaut, on Mar 19 2009, 06:20 AM, said:

I did use a predominantly Taq based enzyme- the kit mentioned that any enzyme system which has a higher proportion of Taq is compatible with theirs and I verified with the enzyme providers too and they said its ok with TA cloning. I managed to get just a few colonies when i plated the competent cells directly on to MacConkey agar, I have pinned my hopes on reviving a culture and then getting it transformed with CaCl? What wud be my odds for that?

As for the time gap b/w pcr and cloning, the first time around I cleaned up the PCR product and stored it at -20 for a few days. The second time, I proceeded directly to cloning after eluting the product from agarose

Please don't mind but rather than asking for your odds on getting positive clone, try to refine your techniques and that will help you in long run.
I wouldn't be surprised if your PCR is sitting in -20°C for days before cloning, for some reason, it hasn't worked for me either. My usual procedure is gel purification of band of interest. No matter if I use Taq-Pfu or plain Taq, I still add A-overhang by incubating with Taq and salts at 70°C for 10 mins and proceed for TA-cloning. This is for cloning your insert of your interest.
Now about your competent cells, use a control. If you have any plasmid lying around pUC19 or similar (preferably same size as your clone) Check how the transformation with your regular lab protocol works out for both. Now that you have a control, you should be able to figure out, if its just the cells or your ligation thats not working.
Hope this helps.

[crackanaut]

Will try that!! thnks for the comments..

Edited by crackanaut, 19 March 2009 - 04:02 AM.

[LP2]

Even though the question has been answered I feel I should mention TOPOXL vector as its never failed me, even when I can barely see the PCR product. It contains a lethal gene that is inactivated by the insert so you get no vector background and Topoisomerase instead of ligase. I use Pfx which has good proof reading and no taq then add 4u Taq at 70oC (as said below) for 5 mins to the PCR tube to add overhangs. I gel extract using either EtBr or crystal violet (gel extracting also removes any template plasmid which may give colonies on the plate) and do the 5  min topo reaction.

[jiajia1987]

LP2, on Mar 20 2009, 01:48 AM, said:

Even though the question has been answered I feel I should mention TOPOXL vector as its never failed me, even when I can barely see the PCR product. It contains a lethal gene that is inactivated by the insert so you get no vector background and Topoisomerase instead of ligase. I use Pfx which has good proof reading and no taq then add 4u Taq at 70oC (as said below) for 5 mins to the PCR tube to add overhangs. I gel extract using either EtBr or crystal violet (gel extracting also removes any template plasmid which may give colonies on the plate) and do the 5  min topo reaction.

Would you mind explaining the words in bold? do you mean that other than your desired PCR products, there were other stuffs that ligated to the vector?

[crackanaut]

the kit recommends a certain concentration of pcr products for ideal vector-product ligation.. i think lp2 meant that he got the req'd reacn even when the eluted pcr product was of low conc..

[LP2]

In response to to the above post by jiajia1987. There were two instances of this.

1) when I loaded 40ul of PCR reaction onto a gel I could just see a faint but discreet band at the desired size. I then used the remaining mix to do the TOPO reaction and got lots of colonies. I screened them and found that they were all correct.

2) When looking into virus splicing patterns I used a sequence specific oligo and an oligoT to make cDNA of the region of interest.  I amplified it for about 5 cycles and TOPO cloned it. This gave colonies, some of which contained junk but 4 or 5 had sequence which told me that my splice site and polyA site were working. (i'm sure there are better ways of doing it but I just used the materials that came to hand and was confident with the result).

Hope this is clearer

Edited by LP2, 22 March 2009 - 07:38 AM.