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DNA ligation and trasformation

Cloning

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6 replies to this topic

#1 ahmadfathi

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Posted 06 February 2013 - 06:50 PM

hello, i have performed ligation for my sample. the concentration of my vector is 8 ng/ul while insert is 15 ng/ul. i used 1:3 ratio of vector insert where insert + vector= 100ng..this sample done in 4,16,23 and 25oC. The ligation product then stored in -20oC and 4 ul of ligation product then used for transformation on dh5a. however i got no cloning culture. i used invitrogen t4 ligase..is there any problems in my methods?

#2 littlepumpkin

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Posted 06 February 2013 - 08:47 PM

You can try to dilute your ligation reaction (after it's done) before doing the transformation. I usually dilute my 5 ul rxn to 25 ul using sterile dd H2O then transform 4 ul. It can lower the salt concentration and increase the yield. Btw, how big is your insert?

#3 neuron

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Posted 06 February 2013 - 10:03 PM

We do ligation at 16 degrees and it works fine. The ratio of insert : vector depends on what vector we are taking. We also use T4 ligase ..so method should be fine.

#4 phage434

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Posted 07 February 2013 - 05:51 AM

Usually the problem is in DNA or the competent cells, not the ligation. Have you measured the competence of your cells? How are your DNA parts being prepared?

#5 neuron

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Posted 07 February 2013 - 08:33 PM

some information about ligation-

http://www.addgene.o...s/DNA_ligation/

#6 Lisa Hsu

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Posted 08 February 2013 - 03:53 AM

Is it a high copy number vector ?
Your ligation sounds fine to me :D
Is it because your insert DNA too toxic for the cell because of the high copy number vector?
It's just a thought Posted Image

#7 ahmadfathi

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Posted 11 February 2013 - 07:22 PM

tq..
littlepumpkin:

Is it a high copy number vector ?
Your ligation sounds fine to me Posted Image
Is it because your insert DNA too toxic for the cell because of the high copy number vector?
It's just a thought Posted Image

what you mean by toxic?
i really need a clearance, i'm still a newbie in this area
my insert size is 1.5k bp..





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