Kev (abr. Kevin)
Irish, Gaelic orgin. Caoimhin.
1. adorable. 2. gentle one

Walkabout (walk-a-bout)
1 (in Oz) - a walk in the Outback by Aborigines that lasts for an indefinite amount of time. 2 (chiefly British) - an informal stroll among a crowd conducted by an important visitor e.g. a monarch. 3 a walking trip.

Friday, 24 August 2007

Problems with work.... again!

Haiz... Been having problem with my cloning work again... and I find that I doubt myself very often... It's like during the experiment I need to add e.g. 20uL of buffer into the reaction, and 10 to 15 seconds after adding it, I will start to question myself if I've added it, and if I added the correct amount. I would start to check my pipette if the correct amount is adjusted etc... I tried to recall, but somehow I just can't say for sure if I really did the correct thing. Sometimes, I just tell myself that I'm just scaring myself, and move on to continue the experiment... all the while enduring the distress of wondering if I did the thing for the experiment! And then I wrote an email to the technical support for Invitrogen, asking them why my reaction couldn't work...

attached email------
Hi, I had recently bought a pET101-DTOPO kit 3 weeks ago, and had problems cloning a 1035bp gene within the vector. Foremost, I did a negative ligation control (without insert) for 30 minutes at 23 degrees, and transformed it into the TOP10 cells. About 60-80 colonies grew on an ampicillin (100ug/mL) plate, after spreading ~250uL of the cells.

My recombinant ligation reaction was at 23 degrees for 30 minutes as well, and spread 250uL of the cells onto an ampicillin plate as well. There were about twice as much colonies as the negative control plates. I screened 12 colonies with colony PCR using T7 and T7 reverse primers only to find that all 12 colonies were absent for my 1035bp insert. As such it seems there is a problem with self-ligation of the pET101 vectors. (I had used a molar ratio of 1:1 for ligation, as suggested in the protocol; Blunt end PCR product is generated with Pwo DNA polymerase with a CACC 5'end forward primer.)

qn) How come there is so much self-ligation of the vector? I suppose this isn't normal, is it? What can be done to reduce such self-ligation, or that there is a problem with the vector? I am simply not getting any recombinants at all, as it turned out that all the colonies that I had screened have a self-ligated vector!


Princess Eileen said...

Haha, Kev such scientific knowledge not many ppl can digest. It's been 2+ years since I left this field... Probably you didnt cut it at the right point or it just didnt get inserted. Or maybe the company give you rejected products!!!

KeV's wAlKAbOuT said...

wow... I didn't expect anyone to understand this post... since this is related to my work, and not many ppl are in this line...

And wow! ur a science student as well huh?!

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De Pianist said...

hehe..paiseh,i don't really understand this post.hehe..btw,what is your job anyway?

bokjae said...

Hi kev, its all Greek to me! Sorry unable to help but just want to encourage you Press On! Success is made of stuffs like these! hehe!

Nicole Tan said...

my god that has got to be like the most scintific post I've ever read anywhere!!!! haha but

1) use another ligating kit!!!! invitrogen one sucks (based on what I've read)....I usually use the one from roche and it works perfectly

2) if you got loads of time, screen all your colonies...who knows you might actually get one that is right...after all I presume you just need one measly one to work right???

3) try changing to all new reagents and buffers..and I'm not sure if you use CIP in your reaction....but play around with that concentration because that is quite important step...

and if it still doesn't work, go pray to the lab god!! :D

Princess Eileen said...

Yes, biotech student..... once upon a time...

Anna said...

In this moment I have the same problem with pET 101. None of my colonies have the insert.
Did you find a solution! If you did, please, tell me!

KeV's wAlKAbOuT said...

Hey Anna, I managed to get my clones finally... didn't manage to circumvent the problem though... solved it by brute force - screening (with colony PCR)over 100 colonies b4 I get one positive clone.

Hope it helps! =)

annadogui said...

I tried brute force too, and I get 2 positive clones from 72 screening.


KeV's wAlKAbOuT said...

Hey Anna, congrats!

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