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och

Member Since 11 Jul 2012
Offline Last Active Sep 28 2012 10:42 AM

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Nayeem991
11 Jul 2012 - 20:49

#137532 Sub-cloning and transformation

Posted Nayeem991 on 12 July 2012 - 10:11 PM

och, on 12 July 2012 - 03:01 PM, 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 (amp^r), 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 phage434wanted to mean it. if you want to use the digested product without purification, in that case you must inactivate the enzyme by heat treatment (65⁰c for 10-15 mins) but don't use EDTA. usually 1:3 vector to insert ration is used in ligation. If you avoid gel run, you have to increase the amount of insert. It could be 1:10. In that case you may increase the final volume of your ligation mixture. You can make it 20 instead of 10.
Best of luck

#137453 Sub-cloning and transformation

Posted Nayeem991 on 12 July 2012 - 02:46 AM

och, on 11 July 2012 - 08:54 PM, said:

@ 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

Do you use whole 10 ul of your ligation mixture for transformation? You can use whole to increase the chance. you should check the efficiency of your competent cell by transforming undigested plasmid. Try again with controls (as i mentioned earlier), i hope you will be able to identify your problem. At the same time you can maintain following caution while preparing the ligation mixture.... add ligation buffer after vigorous vortexing (make sure your buffer is completely thawed and there is no undissolved residues after vortexing). Take the ligase from the surface (do not dip the tip into the enzyme). I don't know but i think these might help to overcome your problem. At least you would be able to identify the problematic step. Best of luck.