Primary cells and cell lines:
All cell lines are to be maintained by the person who grows them, including weekends and holidays. If you can’t be around on specific days to maintain your cell line, please ask someone else in the lab to do this for you rather than letting the line die off.
STERILITY AND CARE are of utmost importance when dealing with cell lines. Please spray down the biosafety hood with 70% ethanol after every procedure, even the most minor one. Always use gloves when working in the biosafety hood. Hood #1 in human cell culture room is meant for handling of primary human cells received by the lab. Please DO NOT use this hood for any other cell types. It is also a good idea to use double gloves especially when handling primary human cell samples. Remove and dispose off all biological waste into designated double bagged biological waste containers and store at -20°C till incineration. Use the freezer compartment on top of the refrigerators for storage of biological waste only. These frost-free freezer compartments should NOT be used for storage of lab reagents / enzymes.
The 37°C water bath chamber near the tissue culture room is for warming mammalian cell culture media ONLY. You can change the one on the left to whatever temperature you want. The water in all waterbaths must be distilled water, supplemented with 0.5 ml of benzalkonium chloride per liter. It is everyone’s responsibility to take care that the water levels are maintained and the waterbaths are cleaned on rotation every month.
IT IS ABSOLUTELY IMPERATIVE that all enzymes should be kept at –20° C at all times. When you use an enzyme, place it in the –20° C freezer block, which is next to the enzyme blocks. Take this block to your bench and aliquot the enzyme from it. NEVER, EVER place any enzyme on wet ice. The only exception to this is alkaline phosphatase, which is kept at 4°C and may be placed on ice.
1. Sequence: if a plasmid was constructed by insertion of an oligonucleotide or a PCR product, the entire inserted fragment must be sequenced to ensure that no sequence errors were introduced.
2. Restriction mapping: if a plasmid was constructed by subcloning a previously sequenced insert from another plasmid, then sequencing is not necessary. Please be sure that you have mapped the plasmid by cutting with a variety of enzymes and have obtained the anticipated fragments. A limited restriction mapping is ALWAYS necessary after maxi-prepping any plasmid.
Storage of plasmids: Maxi-prepped plasmids must be stored in TE at either 4 C or –20 C. If you choose to store cut plasmids (after restriction digestion), these must ALWAYS be stored at –20 C, and for no longer than one month (linearized DNA fragments are subject to exonuclease digestion, and their ligation potential decreases dramatically with storage time).