Sub-cloning and transformation - (Jul/11/2012 )

Hi all,

Please I'm having some very serious problems on my project. What I am trying to do is to:
1. extract an EGFP gene from pSAT1-EGFP-C1 (4553 bp) and clone it onto the binary vector, pPZP-RCS2-ocs-bar-R1 (8637 bp)and then transform it with competent cells.
2. Later on, I will use it to clone some genes from tomato plants by silencing usin VIGS.

But the problem I am having now is in the first step. I havn't been able to clone the EGFP gene onto the binary vector. The protocol I have been using is:
For the vector
(a) plasmid extraction of vector (plasmid conc. is usually high)
( enzyme digestion of vector with Asc1 enzyme (I get one band after cutting)
© agarose gel purification of vector
(d) measurement of vector DNA concentration using nanodrop (sometimes I get low conc.)

For the insert
(a) plasmid extraction of insert (plasmid conc. is usually high)
( enzyme digestion of insert also with Asc1 enzyme (I get 2 bands after cutting, then I use the lower band, with size of like 1600bp, for gel purification)
© agarose gel purification of insert
(d) measurement of insert DNA conc. (also I get very low conc. of DNA)

Then I go ahead with ligation of the vector and insert by adding 4ul of insert to 1ul or 2ul of vector; 1ul of T4 DNA ligase; 1ul of T4 DNA ligase buffer; and add up with ddH2O in a 10ul reaction and incubate @ 16oC overnight.

After that, I then go ahead with transformation using competent cells that were ordered and tested. I grow the ligation mix on spectinomycin selective media since the vector has spectinomycin resistance for 16 hours. But at the end, I will either: not see any growth; or see some king of very tiny colonies that won't grow on LB media.

Please, I really need the help of this forum to know what the prob is. I have been stuck here for several months now. I will really appreciate all kinds of opinions on this.

Hi, I think there is prob in the ligation step. It might be because of the ligase buffer or ligase. Did you check the efficiency of your ligase? You can prepare a reaction mix without the insert and transform that into the competent cell. All the condition should be same, just use ddH2O instead of insert. If you find no colonies yet, i think you should change your ligase and ligase buffer and try it again.

Start by making sure you can transform your vector and grow it on your plates. Does the EGFP source plasmid also have spec resistance? If not, you can eliminate gel purification entirely, which I would strongly recommend. Gel purification has low yield and can often damage DNA with UV exposure.

@ Nayeem991... Thanks for your reply. In fact I a new ligase and ligase buffer before doing the last ligation. But still the transformation still failed. Anyway, I will try your advise and check if the ligase and buffer are still in good condition.

@ Och: You can also try to transform a small amount (may be 1 ul or even more less) of undigested binary vector. Then you would be able to understand whether there is any problem with the transformation step or the competent cell. In which bacterial strain you are trying to transform? If you could tell me the the transformation protocol you followed, i could suggest you any alternative... best of luck.

@ phage434... I appreciate your response. Can you please explain your second statement further. The EGFP source plasmid has an ampicillin resistance. i will like to know why you suggesting eliminating gel purification totally. Thanks a lot.

@ Nayeem991.. I am trying to transform into E.coli. below is the transformation protocol I have been using. I will also appreciate if you will explain the ligation ratio of vector to insert to me. As in, how do I calculate the different amounts of each to mix in a ligation mix. What I have been doing is to add more insert than vector. Thanks a lot.


1. Put 50ul aliquot of competent cell each into 3 tubes.
2. Put on ice immediately and thaw
3. Take 2ul ligation product and put into competent cell
4. Mix gently by pipetting
5. Let sit on ice for 45minutes
6. Heat shock at 42^oC for 2 minutes
7. Put back on ice and incubate for 30minutes
8. Add 500ul SOC into tube
9. Shake at 37^oC, 100rpm for 1 hour
10. Pour 100/200ul bacteria on LB + spc(50mg/l)
11. Incubate at 37^oC overnight

och on Thu Jul 12 04:54:57 2012 said:

Thanks Nayeem991. I will put all your suggestions into use and reply back to you.

@phage434. I now understand your point on the EGFP gene. Now, should I also gel purify the plasmid vector or not?

Thanks man.