BSM

Hey everyone, I want to perform in-situ hybridization on mRNA from cells that already express GFP and RFP. Will expression of these proteins interfere with the TSA fluorescence reaction that occurs during ISH? Is this why proteinase K is sometimes used so it destroys the fluorescing proteins?

Thanks!

Hey everyone,

I plan on performing a nucleofection on primary neural progenitor cells derived from IPSCs so I can label them with GFP and generate a stable line. The only vectors I have found that are compatible are for lentiviral transductions such as this one: http://www.addgene.o.../sequence/8617/

I plan on linearizing the GFP + puromycin region then performing the transfection. Do I need to add in PolyA signals downstream of GFP and puromycin in order to get expression to generate stable clones? Do any of you know of any other plasmids on addgene which would have GFP + a selectable marker that are not designed for lentiviral work?

Thanks

I want to have stable GFP expression in my mouse IPSCs and embryonic stem cells and was looking for the method which would result in the greatest efficiency. I have heard that lentiviral packaging vectors may offer the best results. Would I just use a control vector expressing GFP which every manufacturer carries and see if I can generate stable clones. Should I use site directed incorporation of a GFP construct?

Thanks

I was looking to perform a sequential double digest with AseI and BssHII restriction enzymes to isolate my linear construct that will be used for downstream transgenic mouse applications. I need about 60ug of cut plasmid.

AseI works in NEB buffer 3 @ 37c and BssHII works best at 50c in NEB buffer 3. I was 1st going to perform a pilot run with a small amount of DNA and incubate with BssHII 1st at 50c for about 2 hours, heat inactivate at 80c for 20 minutes then add AseI after the temperature has adjusted back to 37 and incubate 2-3 hours at 37c then heat inactivate at 65c for 20 minutes. The resulting digest should give me 5 fragments, the largest being the one I need.

My question is this, what is the optimal enzyme to DNA ratio to use for a digest with 60ug plasmid and at what total volume? My plasmid concentration is at 1ug/ul. In addition, is it necessary to CIP treat?

Thanks!

I am about to send my plasmid + insert in for sequencing and I was testing out a couple of my plasmid specific primers. The amplicon spans my insert, a tetracycline responsive element (TRE)/minimal CMV promoter and part of the EGFP gene. The amplicon should be around 900bp in length, but when I run the PCR product on a gel it shows up in the 450bp region. For digestion, if I linearize the construct it shows up as the proper size, and if I digest out the insert it shows up as the proper size. However, if I digest out the region that spans my primers, I get a smear on the gel.

My first guess would be that this region forms a strong secondary structure. The tetracycline responsive element is composed of bidirectional nucleotide repeats. If I take just the TRE region and use IDTs hairpin analyzer I get hits with delta G's of -25kcal/mol, Tms of 40c, delta Hs of -530kcal/mol and delta s's of -1680kcal/mol

This encompasses about a 250bp region of the 900bp PCR product. Could this be my problem?