Hi there, i need experts help for my experiment.
I need to ligate 3 pcr fragments, and clone them into a vector.
So i have fragment A, 500bp, fragment B, 800bp and fragment C, 489 bp
i need them to be in this arrangement
PCR of all the fragments have no problems at all.
Next, i start with the ligation of A to B
I column purified them after PCR, and digest them in EcoR1, overnight.
Column purified them again, and ligate them overnight.
i diluted them 100X, and run PCR with Forward primer of A and Reverse primer of B
And i get a nice band at 1300bp.
Next, i column purified AB, digest AB and C with Spe1 overnight, column purified and finally ligate them. Same as what i did for AB.
Here where the problems come....
all i get is smearing, a bright smearing originated from the well.
But i did see a band near to 2000bp (my expected band)
So i proceed with gel extraction, cloning and transformation, but didnt get any colony at all (did it 3 times)
My boss said that i cannot gel extraction it , because the band is too smearing.
He asked me to ligate again, and run the ligation mixture in gel, and visualize them.
But all i can see is smearing, can someone tell me what happen here? i thought i should see bands.
Anyway, i start again my ligation, using new ligase, new ddH2O, new diluted primers, did a new pcr of ABC, and the smearing is gone but the band which i had seen at 2Kb is now disappear along with the smears. All i got is a bands at 500bp, 800bp and some bands under 1000bp.
What happen here?
My hypothesis is, i have a contamination is one of my ligation ingredients, and it seems that my digestion of AB and C is not complete. The bands i saw is just ligation of C with C...
I start a new digestion of AB and C and will ligate them and pcr them tomorrow,
can someone help me?