Problem relation to ligation and transformation
Tom Chang
Monday, 14 September 2009 08:44 UTC
Hello everyone:
My name is Tom, this is my first time join this discussion group.
Last few weeks I had tried to construct a new silencing vector through digested both my backbone binery vector (pJK4) and shuttle vector (which contain the constructed invert repeat) with Sac1/Xho1 and performed overnight ligation by using T4 DNA ligase. This ligated silencing vector was then transformed into E. coli DH5a competent cell by using both heat shock and electroporation followed the invitrogen instruction manual.
However, there are NO colony growth on LB+chloramphenical (10ug/mL) plates. I had tried many methods including different vector molar ratios, different ligation temperature or different DNA:cell ratio. I had done positive control (pJK4 empty vector only) and there are herps of colonies.
So could anyone give me some suggestion about what’s might be wrong relate to my ligation/transformants or are there any new methods worth to try?
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Replies
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Hi Tom,
I cant say accurately, but you can check both the enzymes company(SIB/NEB) and digest with its own company buffer( if the enzyme is from NEB use NEB recommended buffer ) and for long time (2 to 3 hrs) and also phosphatase the vector after digestion. Sometimes when the cloning is not working phosphatasing the vector works.
Best wishes,
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How big (or small, rather) is the insert, and what vector:insert ratios did you try? Sometimes, if it’s really small, it’s just really really hard to ligate, and you may only get one colony. (That’s okay, you only need one, but it’s so hard to miss).
I ask, because inverted repeats for silencing are pretty small, but you can possibly cut out a bigger piece (and maybe do some creative cutting/pasting through intermediate stages with bigger fragments to reach the vector you need). Have you made other silencing vectors like this? We used to have a standard protocol for something where eventually the only variable was the actual sequence, but all the bits were always the same size, so technically all ligations are the same. Is that what this is? You haven’t done a similar type of ligation before that did work? -
Hello:
Is Tom, thanks for replies. The vector is ~9000bps, the fragment is ~3000bps. Previously I had tried different molar ratio including 1:1, 1:3, and 1:5. I had also tried SAP but didn’t work.
My experimental is similar to the work performed by (Fitzgerald et al., 2004). The only binary vector currently available (in our lab)is pJK4, which contain single Sac1/Xho1 site and T-border for AMT. The organism I am working with is difficult to use PEG or biolistic transformation.
Currently I am trying to find the new vectors and waiting for the sequencing results.
If you know any good vector or methods, could you please post it to me?
my webmail address is tcha163@aucklanduni.ac.nzThanks
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Hi Tom,
I would suggest the following steps before you setup the ligation.
1. Ensure both the enzymes are cutting in your vector back bone. You may have to carry out single cuts with SacI and XhoI individually and ensure that they linearize your vector.
2. I am not sure of you are gel eluting your insert before setting up the ligation, if not, this is not a bad thing to perform.
3. To ensure your ligaase and the buffer is fine, perform a mock ligation by digesting the vector with a single enzyme, ligate and transform. If the reagents are fine, you should get lots of colonies.
4. This might be a silly Q, but are you using the right antibiotics for selection?
5. The final plasmid size is >10 KB, may be you can try Stbl3 bacterial strain which is known to hold large plasmids.
All the best.
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Hello:
Thanks for your great help. I have completed this project !!
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