「DNA cloning tips」の編集履歴（バックアップ）一覧はこちら
DNA cloning tips - (2010/06/30 (水) 13:45:48) の１つ前との変更点
追加された行は緑色になります。
削除された行は赤色になります。
I did ligation. However uncountable many colony comes up even in a control plate.
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.
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.
3. If there is not containing Amp, all of colonies are gonna come up.
I am gonna contain Amp in the plate in advance.
30/Jun/2010:
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.
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.
3. If there is not containing Amp, all of colonies are gonna come up.
I am gonna contain Amp in the plate in advance.
