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ligation and transformation


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#1 drsarod

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Posted 23 February 2009 - 07:55 AM

Hi,
I have been trying to do insertional inactivation using a Cat cassette. For this I used Topo vector for the initial cloning, picked clones from them and then digested my clone and also the pcr amplified CAT cassette with the same enzyme (BbsI) and then i am trying to ligate the digested CAT and vector+ insert. But unfortunately i didnt have any transformants (LB + amp and LB + amp+ cat). But I did also use just lb plates without any antibiotics and there my DH5a cells grew. SO I dont know where I am going wrong. ANy suggestiosn??? I have tried the controls for transformation that come with the kit and they work. I am totally stumped!!!!!!!!!

#2 memo

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Posted 04 March 2009 - 05:29 AM

Hi,
I have been trying to do insertional inactivation using a Cat cassette. For this I used Topo vector for the initial cloning, picked clones from them and then digested my clone and also the pcr amplified CAT cassette with the same enzyme (BbsI) and then i am trying to ligate the digested CAT and vector+ insert. But unfortunately i didnt have any transformants (LB + amp and LB + amp+ cat). But I did also use just lb plates without any antibiotics and there my DH5a cells grew. SO I dont know where I am going wrong. ANy suggestiosn??? I have tried the controls for transformation that come with the kit and they work. I am totally stumped!!!!!!!!!


Hi, it seems that you transformation efficiency is too low or you ligase isn't active anymore. Do you have a positive control for your ligation as the problem does not concern your competent bacteria?
I am not sure if you use a ligation control within the kit or a whole plasmid?
Maybe you can use a transformation enhancer to gain more colony forming units?

#3 hanming86

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Posted 04 March 2009 - 06:00 AM

Could you tell me what Topo vector that you're using??

pCR2.1-TOPO vector doesn't contain this BbsI restriction site.
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#4 drsarod

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Posted 17 March 2009 - 07:00 AM

Hi,
I have been trying to do insertional inactivation using a Cat cassette. For this I used Topo vector for the initial cloning, picked clones from them and then digested my clone and also the pcr amplified CAT cassette with the same enzyme (BbsI) and then i am trying to ligate the digested CAT and vector+ insert. But unfortunately i didnt have any transformants (LB + amp and LB + amp+ cat). But I did also use just lb plates without any antibiotics and there my DH5a cells grew. SO I dont know where I am going wrong. ANy suggestiosn??? I have tried the controls for transformation that come with the kit and they work. I am totally stumped!!!!!!!!!


Hi, it seems that you transformation efficiency is too low or you ligase isn't active anymore. Do you have a positive control for your ligation as the problem does not concern your competent bacteria?
I am not sure if you use a ligation control within the kit or a whole plasmid?
Maybe you can use a transformation enhancer to gain more colony forming units?



I actually thought my ligase wasnt working so I bought new enzyme and I also did the positive controls for the transformation and ligation. I got the right reuslts with the positive controls. So I am not sure whats happening here!! totally stuck and stressed out

#5 drsarod

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Posted 17 March 2009 - 07:09 AM

Could you tell me what Topo vector that you're using??

pCR2.1-TOPO vector doesn't contain this BbsI restriction site.


Yes the vector doesnt conatin the site for the enzyme that is why I picked it. It will only cut my insert!!!

#6 memo

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Posted 17 March 2009 - 07:20 AM

Hi,
I have been trying to do insertional inactivation using a Cat cassette. For this I used Topo vector for the initial cloning, picked clones from them and then digested my clone and also the pcr amplified CAT cassette with the same enzyme (BbsI) and then i am trying to ligate the digested CAT and vector+ insert. But unfortunately i didnt have any transformants (LB + amp and LB + amp+ cat). But I did also use just lb plates without any antibiotics and there my DH5a cells grew. SO I dont know where I am going wrong. ANy suggestiosn??? I have tried the controls for transformation that come with the kit and they work. I am totally stumped!!!!!!!!!


Hi, it seems that you transformation efficiency is too low or you ligase isn't active anymore. Do you have a positive control for your ligation as the problem does not concern your competent bacteria?
I am not sure if you use a ligation control within the kit or a whole plasmid?
Maybe you can use a transformation enhancer to gain more colony forming units?



I actually thought my ligase wasnt working so I bought new enzyme and I also did the positive controls for the transformation and ligation. I got the right reuslts with the positive controls. So I am not sure whats happening here!! totally stuck and stressed out


So your ligase isn't the problem but your clones will not transform! Is it possible your clones expressing in DH5a and give toxic reactions?

#7 Bioalla

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Posted 09 August 2011 - 05:05 AM

Hi!
I got the same problems. For my Bachelor Thesis I'm cloning LHCII-Mutants:
I did a ligation an wanted to transform it to DH5a, but it didnt work out. So I tried it with a PUC vector to make sure that the competence was right, and it was. Then I transformed the ligation in JM101-cells (parallel to the transformation in DH5a) and I had many colonies. Has anyone an idea, what happened?
(I'm not really aware of the differences between DH5a and JM101)
It was always worked with DH5a in my lab and it worked well, but now many people have problems...

#8 szanyi24

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Posted 17 August 2011 - 03:43 PM

Hi, currenly working with an insert (60 bp) and try to ligate this into pgex-6p-1 (size is around 4900bp), but still can't get the clone.

if i digest the pgex-6p-1 with Ecor1 or BamH1 after than I can close it up, with the T4 ligase on 14°C (3-4h), so it's working, but if I digest the little insert with BamH1 and Ecor1, and try to ligate to the pgex, it is not working.
I made an insert with sticky end (all of two ends were Ecor1) I digest the vector with Ecor1, and try to ligate, but I could not that.

my ligation:
3ul pcr product
1ul vector
3ul t4 ligase
1ul 10x ligase buffer

total volume: 10ul, 14C, 3h

With the transformation everithing is okay, because I gave positive controll (with Ecor1 digest vector ligation) and negative controll (just the cutting vector).

Please if you can suggest something, it would be very good.




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