ligation problem

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ajose
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ligation problem

Post by ajose » Thu Feb 11, 2010 6:40 pm

hi everyone

I have been trying to ligate a 5.5k insert to 3 k vector. the vector is linearised with EcorI and the insert is a EcorI fragment. I ligate then in the ratio 1:3 vector is to insert and transformed. i could get only self ligated plasmids.

I was confused whether my vector was not linearised. so i just run the gel for my vector and purified it . the next time when i ligated i didnot get any colonies not even self ligated.

Now I was doubting my ligase... so tried T4 ligase and Takara Ligase kit. This time I ligated
1. Vector EcoRI digest (heat inactivated & ETOH Purified) + Insert EcoRI digest gel purified (1:3)
2. Vector EcoRI digest (Gel purified & ETOH Purified) + Insert EcoRI digest gel purified (1:3) using both the kits.

To my surprise I could get colonies for the heat inactivated vector with both the kits.. I wonder why gel purified ones not giving colonies....

After ligation I have checked by running the gel...I could see bands for both the ligations using both the kits...I am very much confused with the results...

CAN ANYONE HELP ME OUT TO SORT THE PROBLEM....I AM VERY MUCH NEW TO CLONING

THANKS

Ajose

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