20.109(F15): TA notes for module 2

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Long overdue for a revision

General notes

Key preparation:

  • Lots of wild-type inverse pericam plasmid (in the pRSET vector) must be available.
  • Need to streak out DE3 and DE3/IPC(wild-type) from frozen stocks in advance of liquid culture setup.
    • To be most careful, may want to freshly transform DE3 with pRSET-IPC miniprep.
    • DE3 in collection is NB301/AB2
    • DE3 w/IPC is NB303/AB4

Scheme: each pair of students will make one IPC mutant.

Daily notes

Day 1

Materials required:

  • Primers for mutagenesis, forward and reverse, premixed at 10 μM.</font color>
  • pRSET-IPC at 25 ng/μL (1 μL per group)
  • NEB 5α competent cells (one 50-μL aliquot per team)
  • Q5 master mix
  • Q5 KLD mix and buffer
  • SOC (1 mL per group)
  • Enzymes (NaeI or NgoMIV, and StyI for diagnostics may need to be ordered as well, if diagnostics digests performed on M2D2.

Preparation

  • PCR tubes on racks obtained from the freezer
  • Prepare a few aliquots of Master Mix for students, plus a control reaction

Reverse primers

Mutation (X#Z) Reverse primer (5' - 3')
D21A AAT GAG AAG GCT TCT TTG AAC TCT GC
E30K ATG GTG CCG TCC CCA TCC
L31R GTG ATG GTG CCG TCC CCA
D57H TCA TTG ATC ATA TCC TGC AAT TC
T78P TTT CTA GCC ATC ATA GTA AGA AAT TC
D93V AAT GCT TCT CGG ATT TCC TC
M123L TCT TCA TCT GTT AAC TTC TCC
D130G TCC CTT ATC ATT TCA TCA ACT TCT TCA TC
D132H TCT GCT TCC CTT ATC ATT TC

At end of day: freeze SDM DNA

How it went:


Preparation for M2D2

  • KLD digestion (by DpnI) of class mutants (1 h)
  • Transform NEB 5α cells, and plate on LB+Amp plates
  • Inoculate all class mutants
  • Mini-prep all mutants (2 colonies per group)
  • Streak BL21(DE3) cells on one LB+Cam plate
  • Finally, at M2D2 J-1, inoculate BL21(DE3) cells in LB+Cam

Day 2

Materials required:

  1. Bacterial transformation
    • LB+Amp+Cam plates - > 1.5 L of LB, separated into 2 flasks for 30 min of autoclave
    • autoclaved glass tubes
    • cultures of competent BL21 cells at OD 0.4-0.6

Day of lab

  • Pre-warm water bath to 42 °C.


How it went:

Day 4

Materials required:

  1. Sub-culture DE3 in the morning.
    • Need 1x5mL tubes per pair, plus two extra to be safe.
    • Typical sub-culture: about 1.5 hours from 0.15 OD to early/mid-log.
    • Last time starting batches at 12:10, 12:30 pm worked well (w/ lecture going till 1:45 pm), but test current crop of cells to double-check growth rate.
  2. Put calcium chloride (prep ~8 mL aliquots) on ice.
  3. LB+Amp/Cam plates
  4. LB broth, Amp, Cam
  5. Miniprep solution aliquots
  6. Sequencing primer thawed and diluted 1:100 TK CHECK/UPDATE
  7. Sterile DI water (200μL aliquots)
  8. Thaw NEB buffers and keep on ice, have enzymes at the ready.
  9. For digests: 12 μL parent IPC and 8 μL M124S miniprep for each group. TK UPDATE, FIGURE OUT AMOUNTS
    • For sense of miniprep stock concentrations, see S10 Day 5 Talk W/F Yellow (E67K), S10 lane 3 in all gels, my notebook (T79P), Fahim's notebook (WT)

Day of Lab (T/W):

  • Quiz (prepared by TA).
  • Keep an eye on DE3 densities before and during lab.
  • Prepare an aliquot each of M124S, T79P, and E67K to be sequenced at Genewiz, so we only need one set of alignment instructions for the students.

Days after Lab (W/R):

  • Store plates in fridge, wrapped in parafilm.
  • On T/W, pick two colonies per mutant to grow O/N in liquid culture (Amp+Cam).
  • Also pick a positive control colony per student plate. (Or just three done by us, known to be correct.)
  • Prepare DE3/WT-IPC in advance for all.
    • Assuming will need to make 1:20 dilutions next time, need at least 2.4 mL of each
    • Prep 3 (3 mL) O/N tubes to be on safe side

How it went:

  • T/R
  • W/F

Day 5

Materials required:

  • Advance prep
    • O/N cultures (see sub-culture notes below)
  • Ice buckets out, per two teams to share.
  • Part 1
    • Put out LB for OD measurements, 10 mL per group plus a couple extra tubes.
    • Sub-cultures
      • Prepare each BL21 mutant candidate, 6 mL per tube.
      • Prepare enough BL21-wild-type and BL21-reference mutants for each pair to have one tube as needed (plus make two extra of WT, and one of each reference mutant).
      • In sum, there should be 4 tubes of BL21 per pair.
      • In the past, ~1:20 dilution initiated between 10:00 and 10:30 am usually worked well. M124S grows somewhat more slowly.
    • Thaw frozen IPTG or prepare fresh (0.1 M stock). IPTG MW = 238.3 g/mol = 0.095 g in 4 mL for 0.1 M. Put out 550 μL per two teams to share.
  • Part 2
    • Thaw digests
    • One-half gel per group and ~ 500 mL TAE buffer per gel available.
      • T/R: Three 1% gels and one 1.3% gel.
      • W/F: Ideal is 2.5 1% gels and 1.5 1.3% gels.
    • Loading dye out at teaching bench, about 4 aliquots (50 μL size) for those groups that did not yet add it on D4 and/or who forgot to prepare uncut samples.
    • Check which groups didn't prepare uncut WT, have miniprep thawed and available in one or a few shared aliquots as needed.
    • 1 Kbp and 100 bp ladders available. Two 100 μL aliquots of the 1 Kbp should suffice for both days.
    • Put out sample sign. at gel bench as reminder.
  • Part 4
    • Colonies out at teaching bench

Day of Lab (R/F):

  • Make sure students turn roller back on!
  • Make sure students measure, then spin down and save at least their -IPTG samples.
  • For recalcitrant +IPTG samples (no color change), continue induction at RT overnight.

Day after Lab (F/Sa):

  • Measure the OD (1:10 dilution), then spin down and freeze any +IPTG samples cultured O/N.
    • Post the OD values to the wiki.

How it went:

  • S13
    • T/R: Lots of samples growing quite slowly compared to previous years. Should start at 10 am instead of 10:30 am for W/F. Possible reasons: chloramphenicol made very fresh -- maybe lower slightly? Ditch antibiotics altogether?
    • W/F:

S10

  • Starting 1:20 sub-culture at 10:30 am (T/R) or even 11:15 am (W/F) gave high log ODs, 1.5 or 1, respectively.
  • More than half the groups had green pellets after 2-2.5 hours of culture.
    • M124S grows more slowly :( Most students had pale yellow pellet, some went with it, some grew O/N. After O/N growth, all pellets were quite green and large.

Day 6

Materials required:

Stuff requiring advance thought/prep: protein purification buffers, BSA standards

  • Part 1: cell lysis, all up at teaching bench
    • BPER (3 mL aliquots, 30 μL 10% BSA and 30 μL protease inhibitors)
      • last-minute prep, as it should be kept at room temp but has enzymes in it
    • Lysis enzyme available on ice up front
  • Part 2: SDS-PAGE advance prep
    • Water (150 μL aliquots)
    • 2X sample buffer available in hood
      • about 100μL group, three 300 μL to share should be fine
      • add 5% β-Me at last minute
  • Part 3A: protein purification (see also googledoc!), in ice buckets at their benches
  • Nu
    • Note: prepare solutions ~15% in excess of needed volume
    • Water, Charge Buffer
    • Binding Buffer, Wash Buffer, and Elution Buffer with protease inhibitors
    • Small BSA aliquots ready.
  • Part 3B: protein purification
  • Part 4: protein concentration, at teaching bench
    • put out closer to end of lab
    • 5X Coomassie stain from Bio-Rad out, three 8 mL aliquots to share (each team needs 2.4 mL)
    • 9.6 mL aliquot of water, one per team (plus an extra)
    • BSA standards --

Day of Lab:

  • Quiz

MOVING ELSEWHERE

  • Transfer gels to fresh water at end of lab and/or next day.
  • Collect all purified protein samples from students and store at 4 °C.

Day after Lab:

  • Transfer gels to water and take pictures.
  • Put up sign in BPEC reserving Day 7 platereader use.

How it went:

  • This is another long day that may need a bit more black-boxing/efficiency work.

Day 7

Materials required:

  1. Pipetting reservoirs - 2 per group
  2. Calcium solutions - 0.5 mL/soln/group

Day of Lab:

  1. SDS-PAGE, gel bench
    • Polyacrylamide gels (1 per pair).
    • TGS buffer (1 L per box)
    • Staining boxes, couple of spatulas
    • Coomassie bottle and 50 mL conical tubes for measuring
    • Distilled water in 1 L bottles
  2. SDS-PAGE, in hood
    • Water baths with boiling chips, turn early on in lab
    • Lid locks
    • Waste bottle for stain
  • Quiz (prepared by TA).
  • Post data to wiki.
  • Instructor teaches the first group of students how to do the multichannel pipetting (avoid bubbles, make sure all tips are actually taking up the same volume, etc.); then TA takes over while instructor is in BPEC for plate reading.

How it went:

  • Protein amounts (fluorescence values) were pretty consistently higher this year than in year's past. In part may be due to using Invitrogen instead of Novagen resin, due to a mix-up; in part may be due to the higher cell ODs for many students. Perhaps in future should aim for high-log rather than mid-log growth, in fact.

Day 8

Materials required:

  1. None: all computer work today.

Day of Lab:

  • Quiz (prepared by TA).

How it went:

  • M124S a much better parallel sample than S101L (run in S09). The change in affinity and cooperativity was dramatic and consistent across the class.
  • The only strange issue is that at very low calcium concentrations the data is noisy rather than simply being a high plateau, or even somewhat consistently starts a little low, then has the high plateau, then proceeds as expected.
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