Experiments This Week
Monday – Finished very carefully making up buffers for the purification; discovered that my Ni-NTA column needed some cleaning; looked up protocols and cleaned it with 0.5M NaOH
Tuesday – made solution of NiCl2 with help of Hill lab for regeneration of Ni-NTA column; made all solutions for regerenation; ended up washing column and stopping just before the EDTA strip (6 ml 7M urea, 15ml ddH2O, 9ml 2% SDS, 3 ml 25% EtOH, 3 ml 50% EtOH, 3 ml 75% EtOH, 15 ml 100% EtOH, 3 ml 75% EtOH, 3 ml 50% EtOH, 3 ml 25% EtOH, 15ml ddH2O). It was looking much more blue after cleaning and should be good for 5 purifications, according to the literature. Purified rtcB using modified Shuman protocol. Concentrated it via Amicon filters – had much less trouble – still had to spin down twice as long. Ran 12% SDS-PAGE gel of boiled and unboiled samples. All samples ran on the gel after staining and were the correct size – 45 kb.
Wednesday – Nanodrop of rtcB (5.2 mg/ml Shuman’s buffer C; 4.3 mg/ml 1x PBS); setup ligation reaction; made stock of GTP and froze aliquots in -20C; poured two 20% sds-page gels and stored in 4C
Thursday – 20% SDS-PAGE gel of ligation reaction – looked terrible and dirty; talked to Samuel Hong, 2nd year in biochemistry – he recommended using proteinase K before loading, and loading in formamide/EDTA (stop soln, also used by Shuman); setup a second ligation reaction and left in heat block o/n.
Friday – filter sterilized gel solution and cleaned gel plates with 2-propanol and sonication ~ 5 min. Ran a 20% SDS-PAGE gel of the second ligation reaction. Stained gel w/ sybr gold that had been stored in 4C 1 hr 40 min and rinsed 20 min. Gel looked much cleaner but data did not make sense.
Purchases for the week = $1.33
Syringe filter, 26 mm, $1.33, 5-31-13
Tentative Schedule for Next Week
Monday – meeting w/ Khalid at 4 p.m.; additional sds-page gel of last week’s reactions, w/o boiling samples
Tuesday – 12% SDS-PAGE gel to make sure protein is still not aggregated. Planning future experiments and look into ordering new substrates from IDT
Wednesday – TBA
Thursday – TBA
Friday – TBA
You can reuse sterile filterers. Always reuse them.
Do not use the mammalian cell culture incubator to store any enzyme reaction overnight. It needs to be completely sterile in there and one must spray down your hands with 70% EtOH before adding or taking out anything out of it.
If you need a 37C reaction for your enzyme, use the water bath with Nanopure water or use the heat block.
Don’t forget controls for your experiments.
It looks as if I have been destroying the 20mer by boiling it – RNA in the middle will hydrolyze at 100C 5 min that I’ve been doing. This is why it’s been disappearing. Will also hydrolyze any RNA on the 10mers.
Potential self-ligation reaction going on in the control sample in presence of Mn(II)Cl2, but it is uncertain at this time.
Very unclear why I got two bands in the phos 1 10mer lane.
SYBR gold has signigicant sequence specificity – binds GC much more than AT. Also, binds ssDNA about ½ less than dsDNA and binds RNA only 7% as much as dsDNA. See the paper: Anal Biochem. 1999 Mar 15;268(2):278-88. “Characterization of SYBR Gold nucleic acid gel stain: a dye optimized for use with 300-nm ultraviolet transilluminators.”