Comments/FAQ

Reagents

Where can I get the reagents for making a mosSCI strain?
My favorite genes are on Chr. II and Chr. IV - do you have other insertion sites?
How can I generate insertion sites that are convenient for my experiments?
Is there anything magic about the ttTi5605 site? Or will any Mos1 insertion site work?

Following Mos1 inserts

I would like to cross the mos1 insert into another genetic background. Do you have a good set of oligos to follow the Mos1 elements?

Getting insertions

I can't get insertions by MosSCI. Grrr - are you making this whole thing up? Or, what am I doing wrong?
How important is the quality of injected DNA?
What is the largest transgene you have inserted?
I can't see expression of my inserted promoter::GFP transgene. What is wrong?
I can't detect germline expression with this promoter that I know should express there. What's wrong?
Nomenclature. How do you designate insertions?
The unc-119 animals are really sick and difficult to inject into. Any advice?

Verifying insertions

I got an insertion! Now, how do I verify it?
How can I know if the full length transgene was inserted?
How can I test if there is a duplicate insertion?
A PCR approach to characterizing MosSCI (developed by Mike Nonet)

Cloning

I can't get the vector pCFJ150 to grow in bacteria. What's wrong?
I am new to Invitrogen's Gateway cloning - which kit should I use?
I am getting a lot of background colonies on my three-way Gateway reaction into pCFJ150. What is wrong?
Can I make my vector smaller by reducing the size of the homology region?

Protocol

The protocol uses three fluorescent co-injection markers? Are they all necessary or can I just use one?
Can I use other co-injection markers than the three mCherry markers?
I am worried about co-inserting unc-119(+) with my gene. Can I use another co-insertion marker?
My transgene is toxic. Can I reduce the concentration in the injection mix and still get insertions?
I've generated transgenic animals with the negative selection (twk-18). However, they are not completely paralyzed when I shift the temperature. Do you think I can get insertions from this strain?
My transgenic line with negative selection was paralyzed at 25C when I tested it. After heat-shock, I see animals that move pretty well with the array. What's going on?
Do you linearize the plasmids before injection?

Negative selection (peel-1)

Can I heat-shock at 37C?
Two hours of heat-shock seems like a very long heat-shock. Why do you do it for so long?
After the heat-shock I still get red animals that move around. Why is that?
Can I heat-shock the animals in a water bath?