February - A slow start - RNA degradation rears its head
Feb 14th, Friday: Ordered splints for the right side of FAM45 substrate
Feb 17th, Monday: 1) Ordered parts for Barnstead Nanopure 2) checked pH of Tris-HCl buffers
Feb 18th, Tuesday: 1) Made new stocks of FAM45mer; pHed a Tris-HCl buffer to pH 8 at 37C
Feb 19th, Wednesday: appt
Feb 20th, Thursday: made up 10x TBE incorrectly
Feb 21st, Friday: 18% urea/SDS-PAGE to rest RNA integrity; it shows degraded; made fresh FAM45 stock
Feb 24th-25th, Tu/W: ?
Feb 27th, Thursday: Checked RNA integrity of fresh FAM stock – it shows degradation
Feb 28th, Friday: ?
March - examining rtcB activity with and without GTP
March 4th, Tuesday: 18% urea/SDS-PAGE testing rtcB activity on DzL7/7 substrates
March 5th, Wednesday: Making up Tris-HCl correctly (tried to do o/n experiment but couldn’t complete it the next day
March 6th, Thursday: 1) DzL7/7 o/n setup for tcB w/ and w/o metal and GTP 2) Ordered the consumables pack for the Nanopure system
March 7th, Friday: 18% urea/SDS-PAGE of o/n reaction, ligation with rtcB compared to ligation of the 3-P RNA substrates
March 10th, Monday: o/n 3-P ligation and DzL7/7 digestion w/ and w/o metal and GTP
March 11th, Tuesday: 18% SDS-PAGE of above reaction; forgot to include 10mer 3-P
March 12th – 20th: 1) Raines’s visit 2) designed new FAM substrate in three parts – did not end up ordering 3) installed the Nanopure system filters
March 21st, Friday: Remaking stock solutions with the new filtered water
March 25th, Tuesday: Designed DNA template and primers for RNA transcription reaction
March 26th, Wednesday: o/n DzL7/7 and DzR8/8 reactions with FAM45 w/ 10x less Dz conc
March 27th, Thursday: rtcB ligation on o/n reactions above (RNA shows degraded)
March 28th, Friday: ?
March 31st, Monday: Remaking Mg and Mn stocks
April - A we'll see how this month goes
April 2nd, Wednesday: 18% SDS-PAGE of FAM45 compared to Fish’s samples and water to determine source of RNA degradation, stained with SYBR gold (gels show RNA is not degraded)
April 3rd, Thursday: