k_undertoe

I recently began using a different product to produce cDNA.  It is a commercial kit with a 2-step protocol, the first using a gDNA elimination step and then proceeds with an RTase from a 'new source' and also a non-specified primer mix, which is said to be good for low abundance transcripts as well as transcript from 5' ends.

SO, I have used the kit and the first time I did not generate ANY amplifiable signal even for my reference genes (GAPDH, RPS19).  I did a side-by-side prep of control fibroblast cDNA using my previous kit, so it was not an issue of bad sample.  Tech support could not come up with any reason it did not work so they sent me a new kit.  I again ran through the protocol, but this time I got signal from everything.  Genes that the fibroblasts do not express came up as positive (Oct4!).  All of my controls are clean- the RT minus control for the fibroblasts shows no amplification, my no-template water only controls come back clear too.  I did no-template controls for both my RT step AND my qPCR step so there is definitely no contamination in the mastermix.

I have no idea where this signal is coming from, considering my RT minus control is clean as well as my no template controls.  Any ideas?

So, I've seen several recommendations when doing phenol:chloroform extraction to first use a saturated phenol:chloroform:isoamyl alcohol solution followed by a second extraction using only chloroform.  I have the correct buffered and saturated PCI solution, but now I am at a loss: what exactly constitutes a 'tris-saturated chloroform' and more importantly how do I make it?  I have now some pure chloroform (well, alcohol added as a stabilizer...) but what do I need to do to it in order to use it in my extraction?  The specific product I have is this: spectrophotometric grade ACS reagent chloroform, Sigma 366919(C-5312)

I am doing a calcium chloride transfection, but I accidentally put too much calcium chloride into the master ! Instead of water, I put in calcium chloride, and added my plasmids at the needed strength.  What I am wondering is, will storing the plasmids in the solution of calcium chloride be detrimental to them?  They are lentiviral plasmids around 5kb.  The solution is made of:

2M calcium chloride, 1.108 mL
About 42 micrograms of plasmid DNA, 73 microliters


Any advice? Are my plasmids okay?  Can I save them?  Can I use the calcium chloride plus plasmids in future transfections?  I wasted a lot of plasmids in the mix and I hate to think of making another maxiprep...

I have made and packaged a viral plasmid into a packaging cell line and now I am trying to name the viral particles that are made from it.  What do I refer to it as?  Like, INSERT NAME + ENVELOPE KIND + CELL RECEPTOR TARGET+ VIRUS KIND + PROMOTER, or what?

The construct I used: pMXs-hNANOG, it is a retroviral plasmid
The packaging cell line: PT67

The promoter for the plasmid is the viral LTR, the packaging cell line provides envelope 10A1, the receptors are GAVL (Pit1) and RAM1 (Pit2).

So, what would the viral particles be named?

Thanks!