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Can s/one help me to solve the problem

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Can s/one help me to solve the problem

Postby huyenkobe » Fri Dec 24, 2010 4:13 am

Hi everyone, I am stuck in this terrible situation for more than 6 months, so I really need your help.I am doing the linker-ligated vector construction. I have a 7 kbase -plasmid with the AsisI cutting site. I design the 3 Myc linker with some nucleotides change in the 5 prime end and 3 prime end so that it can ligate to the Asis I site. I cut my plasmid with AsisI with digested enzyme from BioLab. I annealed my linker with: 2micro S-oligos + 2 micro AS-oligos + 6 micro 5x annealing buffer + 20 micro MillQ water , 100oC 10 mintues, slowly cool down to RT over 30 mns. After that I ligated my cutted plasmid with anneal linker (molar ratio 1/300) with Ligation Mix of Takara during 2hrs. But when transformed to E.Coli, I have no colony.
After several times, I decided to do the self-ligation only. I cut my plasmid, then run gel and extrac the DNA from gel. Then I did the self ligation with 150ng of cut plasmid with Ligation Mix takara during 2 hrs. But I still had no colony. As a control, I did in parallel with uncut plamid and I had so many colonis.
After several months, I recognized that the pH of my MillQ is too high (around 9). I changed the water, and tried the self-ligation one more time. And this time it worked. Then I went forward to do the liker-ligate vector construction. This time I had many colonies. I checked about > 200 colonies but I did not have the linker inside (only the plasmid self ligate). Therefor I decided to repeat cut plasmid and changed the molar ratio (increase molar ratio ). This time I had no colony. And even when I returned to the previous molar ratio, I had nocoly. Now, I try the self-ligation again and no colony at all.
So anyone can tell me what is happening and how to solve it. I already change all buffer with new water. Moreover, I was successful with 3 Flag and 3HA- linker-ligated vector construction without any troubles.
Thank you.
huyenkobe
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