Sub-cloning and transformation - (Jul/11/2012 )
Another thing.... Take three plates and spread whole 500 ul bacteria (~150 ul in each plate) after growing in SOC... Please let me know after performing the experiment. I am eager to know the result.. I am giving you a final outline...
For ligation,
Tube 1: digested and dephosphorylated vector + insert (To get your desired clone)
Tube 2: digested and dephosphorylated vector and no insert (To make sure that your dephosphorylation is ok)
Tube 3: digested vector (not dephosphorylated) and no insert (To check your ligase or buffer is working well)
In addition transform undigested plasmid into the host to check the efficiency of your competent cell.
Regards
Nayeem
Thanks a lot Nayeem991. I will surely let you know when I perform it.
Do you think I can ligate gel purified binary vector DNA(spec resistance) with unpurified insert DNA (Amp resistance) and still get the gene to be cloned onto the binary vector?
och on Thu Jul 12 23:01:45 2012 said:
Thanks a lot Nayeem991. I will surely let you know when I perform it.
Do you think I can ligate gel purified binary vector DNA(spec resistance) with unpurified insert DNA (Amp resistance) and still get the gene to be cloned onto the binary vector?
I wont suggest you to use insert without purification. You can avoid gel run. You can purify the product after digestion. It will spare your DNA from UV exposure. Since the insert size is smaller than the original host vector (ampr), the chances of the insert is higher to be cloned and if there is any undigested vector remain in the digest you wont get it because of spec selection. Did you get my point? I think
Best of luck
So how do I purify without gel run. do you a protocol for that?
Should I also do the same thing to the binary vector?
Thanks a lot.
och on Fri Jul 13 13:50:24 2012 said:
So how do I purify without gel run. do you a protocol for that?
Should I also do the same thing to the binary vector?
Thanks a lot.
which kit do you use for purification? I use promega gel purification kit. The protocol is as same as gel purification. In gel purification... we cut the gel weigh it and add equal volume of membrane binding solution (like.. 100 ul for 100 mg gel) and heat at 65. isnt it?
In case of purification without gel run... add equal volume of membrane binding solution ( 50 ul for your 50 ul digestion mix). If the volume of your digestion mix is 50 ul, you can add 50 ul ddH20 to make it 100 and then add 100 ul membrane binding solution (i always do this thing). then take it to column. the remaining procedure is same as gel purification. If you need not to isolate a particular band this procedure works well. I have done it several times and got very good result.
If you have any query regarding this, please let me know.
And yes i don't suggest you to do the same for binary vector. Because the undigested (if there is any) plasmid will give rise to false result unless you have blue white selection.
Sorry for replying late. I use QIAGEN II purification kit.
ok. I got you. Thanks so much.
best of luck.... 
Do you make your own competent cells? If not, are you following the transformation protocol that comes with them? Two minutes seems like it is unnecessarily long for the heat shock. Reducing to 30-45 seconds should increase your efficiency 2-3x. (http://www.itescam.edu.mx/principal/sylabus/fpdb/recursos/r72068.PDF) Also, every transformation protocol I have seen specifically says NOT to mix the plasmid and bacteria by pipetting up and down, since the competent cells are extremely fragile. A gentle flick or two will do fine. Finally, in my experience, increasing the amount of ligation product you use for transformation to 10uL as Nayeem suggests is likely to substantially reduce the transformation efficiency. I'd stick with 2uL.
I agree with Nayeem though, the first thing you need to do is run some controls, esp. with the uncut vector.