20.109(S09): TA notes for module 1

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.

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).
 * 3) *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 &mu;L of buffer, 1 &mu;L of dNTPs, and 37 &mu;L of water, for a total of 43 &mu;L.
 * See googledoc for total calculations
 * Control rxn. is: 43 &mu;L Master Mix, 2 &mu;L DNA, 1.25 &mu;L each primer, and thus 2.5 &mu;L 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
 * 2) *1% agarose gels, up to 6 groups can fit per gel
 * 3) *TAE buffer
 * 4) *1 Kb ladder
 * 5) Bacterial transformation
 * 6) *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
 * 7) *autoclaved glass tubes
 * 8) *LB broth
 * 9) *1000X ampicillin - central stock per day
 * 10) *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.
 * 2) *Need 1x5mL tubes per pair, plus two extra to be safe.
 * 3) *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.
 * 4) *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.
 * 5) Put calcium chloride (prep ~8 mL aliquots) on ice.
 * 6) LB+Amp/Cam plates
 * 7) LB broth, Amp, Cam
 * 8) Miniprep solution aliquots
 * 9) Sequencing primers thawed and diliuted 1:100
 * 10) Sterile DI water (200&mu;L aliquots)
 * 11) 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.
 * 2) *Last year, ~1:20 dilution initiated between 10:00 and 10:30 am worked well.
 * 3) *This means 4 tubes of mutants per pair.
 * 4) Also sub-culture enough DE3/wild-type and DE3/S101L for each pair to have one tube (plus make two extra).
 * 5) 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
 * 2) *BPER (4 mL aliquots, +40 &mu;L 10% BSA and inhibitors)
 * 3) SDS-PAGE
 * 4) *Polyacrylamide gels (1 per pair).
 * 5) *TGS buffer
 * 6) *2X sample buffer (142.5 &mu;L aliquots, add 7.5 &mu;L &beta;-Me last-minute)
 * 7) *Water (150 &mu;L aliquots)
 * 8) Protein purification (see googledoc)
 * 9) *Note: prepare solutions ~15% in excess of needed volume
 * 10) *Water, Charge Buffer (actually, will probably will buy pre-charged resin this year)
 * 11) *Binding Buffer, Wash Buffer, and Elution Buffer w/protease inhibitors
 * 12) Protein concentration
 * 13) *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 &deg;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).