20.109(S09): TA notes for module 1

From OpenWetWare

Jump to: navigation, search

Contents

General notes

Key preparation:

  • Lots of wild-type inverse pericam plasmid (in the pRSET vector) must be available.
  • Mutant IPC plasmid (S101L) should also be prepared in advance.
  • 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 two protein mutants, and test two candidates per mutant. This year, students will choose only one mutation of their own, and run a 'positive control' mutation in parallel.

Daily Notes

Day 1

Materials required:

  1. None: all work today is computer work.

Day of Lab (T/W):

  • No quiz.
  • Primers for mutagenesis must be ordered right away, rush delivery!
  • Enzymes for diagnostics may need to be ordered as well, check designs.

Day 2

Materials required:

  1. RT water for primer resuspension
  2. Quick-Change SDM kit. 1 reaction per student, plus 1 control reaction, plus spare reagents (ideally).
    • cat # 200519

Day of Lab (T/W):

  • Quiz (prepared by TA)
  • Prepare PCR tubes on racks obtained from the freezer
  • Get primers (forward and reverse) ready just before lab lecture ends
  • Prepare a few aliquots of Master Mix for students, plus a control reaction:
    • Each mutagenesis reaction should have 5 μL of buffer, 1 μL of dNTPs, and 37 μL of water, for a total of 43 μL.
    • See googledoc for total calculations
    • Control rxn. is: 43 μL Master Mix, 2 μL DNA, 1.25 μL each primer, and thus 2.5 μL extra water.
  • Guide journal article discussion (assign figures at beginning of class).
  • At end of day: freeze SDM DNA

How it went:

Day 3

Materials required:

  1. Agarose gel electrophoresis
    • 1% agarose gels, up to 6 groups can fit per gel
    • TAE buffer
    • 1 Kb ladder
  2. Bacterial transformation
    • LB+Amp plates - > 1.5 l of LB requires 45 min of autoclave and is hard to pour, should separate into 2 flasks for 30 min of autoclave
    • autoclaved glass tubes
    • LB broth
    • 1000X ampicillin - central stock per day
    • competent XL1-Blue cells (come with SDM kit)

Day of Lab (R/F):

  • Pre-warm water bath to 42 C, tube racks, etc.
  • Teaching faculty should prepare one positive control plate (in addition to the ones made by the students).

Days after Lab (F/Sa-M/T):

  • On F/Sa, ensure that teaching faculty positive control (pWhitescript) produced colonies and check student plates.
    • Move all plates to fridge for now. Week off until next lab, so wrap plates tightly w/parafilm!
  • On M/T, pluck two colonies per mutant plate, and grow liquid O/N cultures. (Amp only, no Cam yet!)
    • Students with no colonies will be given the alternate candidate from the pilot (Y64D).

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: from OD > 2 to OD = 0.1 may take ~3-5 hours to reach OD = 0.6. Sub-culture to 0.15 or even 0.2 makes for shorter and more predictable lag phase - go with that! Stagger tubes a bit and test after 2-3 h to be safe.
    • Last year starting batches at 12:10, 12:30 pm worked well (w/ lecture going till 1:45 pm), but test cells day before 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 primers thawed and diliuted 1:100
  7. Sterile DI water (200μL aliquots)
  8. Thaw NEB buffers and keep on ice, have enzymes at the ready.

Day of Lab (R/F):

  • Quiz (prepared by TA).
  • Keep an eye on DE3 densities before and during lab.

Days after Lab (F/Sa-M/T):

  • Pick two colonies per mutant to grow O/N in liquid culture (Amp+Cam).
    • Use either the 1X or 10X dilution of cells, depending which has independently accessible colonies.
  • Also pluck DE3/WT-IPC and DE3/S101L
    • 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: Once again, growing for ~ 1.5 hours seemed ideal.

Day 5

Materials required:

  1. Sub-culture each DE3/mutant, 6 mL per tube.
    • Last year, ~1:20 dilution initiated between 10:00 and 10:30 am worked well.
    • This means 4 tubes of mutants per pair.
  2. Also sub-culture enough DE3/wild-type and DE3/S101L for each pair to have one tube (plus make two extra).
  3. 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.

Day of Lab (T/W):

  • Short quiz.
  • Make sure students measure, then spin down and save at least their -IPTG samples.
  • For recalcitrant +IPTG samples (no colour change), continue induction at RT overnight.

Day after Lab (W/R):

  • 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:

  • Starting 1:20 sub-culture at 10:30 am or even a little later (using very fresh LB) was fine.
    • WT and S101L ~ 3 OD, mutants between 2 and 3 OD.
  • More than half the groups had green pellets after 2-2.5 hours of culture.

Day 6

Materials required:

  1. Cell lysis
    • BPER (4 mL aliquots, +40 μL 10% BSA and inhibitors)
  2. SDS-PAGE
    • Polyacrylamide gels (1 per pair).
    • TGS buffer
    • 2X sample buffer (142.5 μL aliquots, add 7.5 μL β-Me last-minute)
    • Water (150 μL aliquots)
  3. Protein purification (see googledoc)
    • Note: prepare solutions ~15% in excess of needed volume
    • Water, Charge Buffer (actually, will probably will buy pre-charged resin this year)
    • Binding Buffer, Wash Buffer, and Elution Buffer w/protease inhibitors
  4. Protein concentration
    • Have 5X Coomassie stain from Bio-Rad, water, tubes ready

Day of Lab:

  • No quiz - a very busy day!
  • 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.

Day 7

Materials required:

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

Day of Lab:

  • Quiz (prepared by TA).
  • Post data to wiki.

Day 8

Materials required:

  1. None: all computer work today.

Day of Lab:

  • Quiz (prepared by TA).
Personal tools