My strange cloning story

It's just a very simple cloning: swap fragment A and B (both ~500bp), link A_up to B_dn, and B_up to A_dn. I begin making this construct with my PI since I just get into my current lab in June. To advance the rate of success, each of us did parallel experiments.

At first we just cut the two ends with KpnI and NheI, and the middle with SgrAI. Ironically each of us tried several times, but still never got the right thing. David think that maybe KpnI, which ends up a 5' overhang, is not a good enzyme for this cloning (although he it seemed fine in the past). He just want to give up and change to our 2nd stratagy: use PCR methods and change restriction sites by primers.

I insist on trying another enzyme instead of KpnI: Acc65I, which gives a 3' overhang. David don't favor trying that at all. So I did that on my own. But it still turned out to be not working. So I just moved along with him. One month already passed here.

David finished ligating the PCRed up and down fragments together, then went to vocation and gave everything to me. It's such a great day that day, I went out to hug every one in the lab, because finally I got the right A_up-B_dn thing! But strangely, none of the white colonies in the B_up-A_dn one works.

I did another set of PCR, digestion, phosphrylation, ligation (3 way including a blunt end), finally 2 out of the 18 colonies seem to work! The sequencing result is exciting but also dissappointing, it's the right swapped sequence, only with 1 PCR error! I picked another set of white colonies, again, 1 PCR error(different PCR reaction)!

I repeated the whole set of experiments again. I picked up all the possible white colonies, 2 out of the 28 got the insert, but still, PCR error again! You can't go to criticize the PCR protocol, because we already perfect it very much. We ran it only 15 cycles to turn down errors. You are just 1 um away from success, and it just seems to be a question of probability. Things are driving you crazy. I believe I am the one that luck tried to avoid.

David come back and repeated the whole thing. Funny enough, it's the same result, you got white colonies, you get 1 out of 18, and the sequencing result tells you 1 PCR error again.

Things beginning to change here. David did something that any molecular scientist can imagine. He went to pick up some blue colonies and give me to do the mini-prep and diagnostic cuts. You know what? The blue colonies got inserts!!!!

Here is our hypothesis:
1. Because our fragments are too short. It's not enough to disturb the LacZ gene to express beta-galactosidase. Thus even though it got inserts, it still form blue colony.
2. In the opposite, for those got inserts but form white colonies, it's because the frame shift in the insert totally disruptted the protein. As a result, actully all the white colonies are the ones with PCR error inside!

Something you can never believe: Blue white selection, blue is right!