Welcome to the Tol2kit blog
This is a forum for discussing the
Tol2kit; the Lawson lab's
related system; and related topics.
The
Tol2kit is a set of constructs that can be used with Invitrogen's multisite Gateway recombination-based cloning system to build expression constructs in a Tol2 transposon backbone (derived from constructs from the
Kawakami lab).
At present each post serves as a topic under which visitors may post comments, either commenting or asking questions. We encourage you to post with your full name (not just your first name) so that we have some idea who is asking.
30 October 2007: There has been a fair amount of spam recently, so I have turned on word verification for comments. You will have to do a "word recognition" task before you can post a comment. Please email me directly if you have any problems with this. --Chi-Bin
2 comments:
Hi Chi Bin,
I did the controls as advised by you.
I did a LR reaction with p5E betaactin + pME-EGFP + p3E-polyA and pDestTol2GG2. The reaction was done in 10 microliter volume and we used the LR clonase II plus enzyme. After Overnight (>16hrs) incubation,5 microliters was transformed into Top10 cells from Invitrogen and 5 microliters was transformed into ultracompetent DH5 alpha prepared by me at the lab.
With top10 cells I got only 2 colonies and both were white and opaque. With DH5 alpha, I got ~20 colonies but all were minute and difficult to characterize as opaque or translucent. I am going to check them for the constructs. I wanted to know if this was as expected....
I did two more reactions to check if the enzyme was working fine and if my construct DNA preparation was fine.
I did two more single fragment LR reactions pME EGFP (from your entry vector collection) + pTolDest - R1R2 (from Lawson Lab) Reaction#2: pME-E1bEGFP (constructed by me) + pTolDest - R1R2 (from Lawson Lab).
In both reactions, I got about 500 colonies for every 5 microliters transformed with Top10 cells. I got even higher number with DH5 alpha cells.
So I suppose the constructs and quality of DNA is good. The enzyme is atleast good for single fragment LR reaction.
Your suggestions and remarks are most welcome...
Thank you
Hi Arul --
Quick thing: I just want to make sure you did the Proteinase K step before transformation; that could definitely affect your results. Also, for your control, I would actually try using one of the IRES constructs as your 3' clone, as p3E-polyA does have a tendency to be less efficient than the IRES constructs (we believe this to be due to the short length (196 bp) of the insert. Finally, you are definitely using 20 femtomoles of each vector?
Kristen
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