DNA cloning tips



I finally have got PIP3FLIMPSD version.
I was very tired..

I started this project from 28May2010.
I finished 25Aug2010. 3 month!!!
What a hell I doing??

The points I improve are... I don't know...
I did the experiments this time carefully.
Of course, I did earlier.

One hard point I got is that I can not cut DNA with enzyme.
Possibly incubation time was short..

In addition, I did enzyme cut for vectors twice during ligation.
And for me, PAP was not so useful...


The resulet was failure...
Why can't I do single ligation?


The result of BAP treatment

Got it!!
A lot of colonies I got without BAP treatment disapeared perfectly even though sample colonies are there.
This data indicates that the colonies in the control plate is due to self-ligation of vector, and is a great step for me. I have new tip to remove background.

I did ligation again.
But a lot of colonies come up in both, ctrl and sample, again.
Normaly thinking is that the vector is not cut enough.
However I carried out enzyme cut for 5 hours.
I can't believe that...

Anyhow I did BAP treatment.
I am gonna compare the samples with or without BAP treatment.

I did ligation. However uncountable many colony comes up even in a control plate.

(Experimental procedure)
I put 400ng cDNA vector.
I put 2microg insert.

I purified both DNAs.

Takara ligation kit ver. 2.1 was used.
I enter 6 microl ligation solution, 5microl insert, 1 microl vector.

on ice for 5 min.
heart shock for 45 sec.
on ice for 5 min.

Add 400 microl SOC buffer and incubated 30min.
plating this solution.

The points I doubt..
1. Exactly KpnI and XhoI works some extent, because the cut insert band is visible. However possibly the ability of cut of enzyme becames lower.
I used KpnI earlier and works well.
So I guess kpnI is gonna fine.
Concern is XhoI.
So what I have to do is to change enzyme.

2. There a monster of colony both in control and insert plate.
I suspect that SOC from Takara is contaminated.
I am gonna use LB buffer I prepared.

Solution: comtamination check.

3. If there is not containing Amp, all of colonies are gonna come up.
I am gonna contain Amp in the plate in advance.

Solution: Possibly the concentration of Amp is low. I am gonna
plate E.coli without amp on the amp plate.

4. Effect of dam methylase
I look up the effect of dam methylase on kpnI and XhoI. But there is no referenece for this. So, It is gonna be no problem.