Seok-Yong said...
Hi,
I'm wondering if it's OK to use a T3 promoter on pDestTol2pA2 to generate an in situ probe upon completing 3-fragment gateway cloning (p5E-CMV/SP6 + pME-gene of interest + p3E-polyA).
...
Just curious! Thanks. SY
AUGUST 30, 2007 10:08 PM
Chi-Bin Chien said...
Hi Seok-Yong,
I would advise against using this T3 promoter to make in situ probe. You would be picking up about 900 bp of vector sequence, which adds a lot of potential for background staining. In particular, you would never want to use this probe on any sort of transgenic fish, because it is likely to detect any transgene made with either Tol2 or with Bluescript-based vectors.
Instead, why not just use pCSDest (#201 from Lawson lab:http://lawsonlab.umassmed.edu/GWDestplasmids.htm) to make a pCS2-like plasmid containing your gene of interest? Then you can use T7 to make antisense probe.
AUGUST 31, 2007 6:37 AM
Seok-Yong said...
Many thanks for swift response. As you suggested, I will generate another construct using pCSDest. Thanks again. SY
AUGUST 31, 2007 1:16 PM
Seok-Yong said...
Hi,
Does anyone have experience with generating DIG-labeled RNA from a gateway vector?
I placed my gene (1.7 kb) on pCSDest and did in vitro transcription (BamHI, T7). The RNA generated was pure, yet the amount was so little. My control plasmid gave me a lot of RNA though. Is it possible that somehow att site could inhibit transcription?
Any input would be appreciated. Thanks. SY
OCTOBER 5, 2007 1:13 PM