I thaw my cells (usually about 1mL) and transfer them in to a Falcon tube containing 10mL of fresh medium. I spin the cells down for 3 mins at about 250g (quite gently) then pipette off about 90% of the supernatant. Then add fresh medium again and transfer this and the cells to a TC flask. The day ...

The most important thing is to ensure that your plates are clean. I use a weak SDS solution, water and then 70% ethanol and then give them a good polish. When pouring the gels I use a pipette and do not blow out the last bit of liquid. Finally, after pouring the separation gel I CAREFULLY add a smal ...

I would also recommend using Invitrogen's TOPO vectors but I'd suggest doing your PCR using a proof-reading polymerase and then ligating into the TOPO-blunt plasmid. But, you said that you already have the PCR product in a pGEM-T plasmid - When you remove it using EcoR I and Sal I it gives you a ...

Just to add that some people in our department are now using temperature sensitive TC plates which enable them to passage macrophages more gently. I don't remember the supplier's details (any ideas HeeHaw?) - just that the plates are quite new and their supply is not high enough to satisfy current d ...

Are the cells you are FACS analysing fixed?

I hope it works out with the new primers. The only other thing I can think of is that certain wells in your PCR block are not getting up to 95 degrees as they should. Could there be dirt/dust etc in some of the wells? I think I'd run some samples on a different PCR machine to rule this out. Good ...

I know this might sound stupid, but are you sure you didn't use un-treated plates by mistake? Are the plates you refer to 10cm petri dishes? (as used for pouring LB plates etc). We have had this problem in the past. The student that stocked the tissue culture lab grabbed a number of packs of petri d ...

You should be able to get 80% transfection efficiencies with home made solutions for calcium phosphate transfections. The quality of the cells is probably the most important factor. They should never be allowed to become over-confluent, so split 1:10 every 3 days.

I agree with Lynn. Your 3.5Kb ORF should be cloned from NcoI to Sal I. It's a little confusing with the numbering as the T7-S-MCS-His-term cassette runs in the reverse orientation to the plasmid's numbering.

Did you say that your "water" control also gave the ladder? Does this control contain everything except the genomic DNA sample? If the water control has no genomic DNA then the bands are due to primer-dimer. You could run the water control with each primer on its own to see whether one o ...