20.109(S08): TA notes for module 1

General notes
Key preparation:


 * In case something goes wrong, spare PCR product should be prepared by TA prior to term.
 * A plan for preparing the high volume of cell culture must be determined well in advance.

Plasmids used:


 * pCX-EGFP:
 * contains full-length EGFP gene
 * Amp resistant
 * f1 ori
 * SV40 ori


 * pCX-NNX:
 * a mock version of pCX-EGFP, contains no EGFP gene
 * Amp resistant
 * f1 ori
 * some altered restriction sites compared to pCX-EGFP, and SV40 ori deleted


 * pCX-EGFP with 3' deletion - still have this in lab, how labeled?

Day 1
Materials required:


 * 1) PCR Master Mix (2.5X), ~ 50 &mu;L per group
 * 2) DI water, prep 100 &mu;L aliquot per group
 * 3) 5 &mu;L aliquots each of pCX-EGFP, D32N-fwd, D32N-rev
 * 4) 2 PCR tubes per group

Keep everything cold!

Day of Lab:


 * Lab orientation quiz (not a TA quiz) will be taken today.
 * Remember to freeze PCR products when they are ready.

Instructor's Bench:
 * Ice bucket
 * PCR Master Mix (taken out of -20C midway through labtime)
 * Sterile H20 in 15mL Falcon
 * Primters for PCR (D32N-fwd: NO47, D32N-rev: NO48, 100 pmol/ul)
 * Template for PCR (pCX-EGFP 100 ng/ul)
 * Beaker with PCR tubes

Prep Work

pCX-EGFP has to be prepared from the glycerol stock. The bacterial stock is NBXXX and a few Qiagen minipreps should be enough for the module (some will also be needed later as a transfection control). You should determine the concentration of the stock. For PCR, dilute stock to about 100 ng/ul.

Other DNA samples that will be needed later in the module come from:
 * NBXXXX    pCX-NNX
 * NB153     pCX-D32N (Xba/RI)
 * NB168     pCX-D3G+Linker -- This plasmid needs to be cut in three ways and each bkb purified!

Check the PCR machine has proper protocol under NK1:

94C 4min

(Repeat 35 times)

94C 1 min

55C 1 min 72C 1 min

72C 10min

4C hold forever

Day 2
Materials required:


 * 1) Qiagen QIAquick PCR purification kit
 * 2) *order # 28104, 50 rxns
 * 3) *1 rxn per group
 * 4) pCX-NNX, 20 &mu;L per group
 * 5) NEB2 Buffer (~5 &mu;L used per group)
 * 6) EcoRI and XbaI enzymes (~2 &mu;L used per group)

Day of Lab:


 * Quiz (prepared by TA).
 * Thaw PCR products and template on ice.

Prep Work

Photocopy pages 232 to 233 from NEB catalog for students to read for next time.

Check STOCKS for TC materials

Filtered DMEM Complete

500mL DMEM (high glucose). 50mL serum (FBS from Atlanta Biological). 5mL P/S/G. 1mL BME. 5mL NEAA

Filtered DMEM Pre-Txn Media

Filtered 500mL DMEM (highglucose). 50mL serum (FBS). 5mL 100XS glutamine. 1mL BME. 5mL NEAA

Day 3
Materials required:


 * 1) Qiagen QIAquick gel extraction kit
 * 2) *2 extractions per group
 * 3) *order # 28704, 50 rxns
 * 4) isopropanol (prep 500 &mu;L aliquots)
 * 5) sterile DI water (prep 500 &mu;L aliquots)
 * 6) loading dye for agarose gel electrophoresis (prep 35 &mu;L aliquots)
 * 7) 1% agarose gels prepared in 0.5X TBE buffer
 * 8) * each group has 10 samples, so requires 10 wells
 * 9) * prepare 1 gel per two groups, but with 2 combs
 * 10) * also prepare 1 more gel (2 combs!), for running purified products
 * 11) Single-enzyme digests - may be prepared on Day 2.
 * 12) *pCX-NNX, XbaI cut only
 * 13) *pCX-NNX, EcoRI cut only
 * 14) TC Room
 * 15) *Razors, Face Masks, Film/Camera, Transilluminator, Spatulas, Kimwips, Eppendorf, Pen, EtBR waste jar
 * 16) Minigel (1%) to check recovery of student's fragments after class

Day of Lab:


 * Quiz (prepared by TA).
 * Run purified products (2 per group) on an agarose gel at the end of the day - post photograph.
 * Freeze DNA at end of day.

Prep Work


 * TC should be ongoing
 * Day 6: 1 60mm dish of MES per person
 * Day 7: 1 24 well dish of MES per group

Post Lab Prep

Between Day 4 and Day 5 you will need to set up 4 LBA overnight cultures/group. Innoculate one with a plug of pCX-NNX and three with bkb+insert plates. Grow 2.5mL at 37C the night before Day 5 lab.

Check stock of enzymes for digest that will be done Day 5

Day 4
Materials required:


 * 1) Retrieve at last minute and keep on cold rack: ligation buffer, T4 ligase
 * 2) 3 M sodium acetate (prep 100 &mu;L aliquots)
 * 3) Yeast tRNA (prep 25 &mu;L aliquots)
 * 4) Cold 100% ethanol (prep 1 mL aliquots)
 * 5) Cold 70% ethanol (prep 3 mL aliquots)
 * 6) Sterile DI water (prep 100 &mu;L aliquots)
 * 7) pCX-EGFP (1 &mu;L/50 ng per group)
 * 8) competent cells
 * 9) * XL1-Blue from Stratagene, cat # ?200249?
 * 10) * 1 tube per group
 * 11) LB+Amp plates, 5 per group + spares
 * 12) * VWR cat. #
 * 13) * may get ~ 40 plates/Liter LB
 * 14) LB liquid medium

Day of Lab:


 * Students Benchtop
 * LB in 15mL conical tube
 * LB+Amp plates (5 per group, so at least 30 per lab)
 * 1mL 3M NaAc
 * Ice buckets with ice
 * 15mL conical tube with 100% EtOH in ice
 * Check spreaders, refill EtOH beakers and alcohol burners


 * Instructors Benchtop
 * Ice bucket with ligation buffer
 * tRNA
 * 2x 50mL conical tube with 70% EtOH
 * pCX-EGFP transformation control
 * Midway through lab, thaw supercomp cells (~400-500ul/group, need 12 tubes of ~200ul each for class of 12)


 * Quiz (prepared by TA).
 * Keep reagents for ligation reactions cold, make available to students as needed.
 * Demonstrate and supervise bacterial transformation protocol.
 * Transform cells with pCX-NNX to get fresh colonies.
 * May be done a week or so in advance, if desired.
 * Can also just try streaking frozen stock, if available.
 * Tomorrow pick colonies for inoculations - 3 candidates per group, plus 1 pCX-NNX.

Day 5
Materials required:

Student Benchtop
 * Liquid cultures (3 candidates, 1 pCX-NNX control per group).
 * Miniprep solutions
 * Soln I (prep 400 &mu;L aliquots)
 * Soln II components (prep 600 &mu;L aliquots, each)
 * Soln III (prep 800 &mu;L aliquots)
 * 100 % ethanol (prep 5 mL aliquots)
 * 70 % ethanol (prep 3 mL aliquots)
 * Sterile DI water (prep 250 &mu;L aliquots)
 * Digests: have all 4 NEB buffers and many restriction enzymes available
 * NEB catalog

Instructor Benchtop
 * Ice bucket
 * Midway through class will need EcoRV, XbaI, BamHI, XhoI and perhaps other enzymes
 * 10X NEB buffers 1, 2, 3, 4 thawed

Ensure DNA is frozen at end of day

Prep Work

Set up liquid cultures the day before! There will be questions on buffer compatibility and anticipated size for fragments. TC will need 12 60mm dish of confluent J1s for next time!!

Day 6
This lab can be chaotic since essentially two labs are running at once. There will be 6 students in the TC facility splitting cells and 6 in the main lab running a gel. Halfway through they will switch.

Long-term preparation:


 * Per student, one 60 mm dish of MES cells near confluence
 * Six 1% agarose gels with EtBr (10 wells/row)

Materials required:


 * 1) Agarose gels
 * 2) *Half of class at a time will be in TC, so can't prepare just 1 gel per two groups
 * 3) *Probably want to prepare 4 gels per 6 groups, but can prepare 1 per group for simplicity
 * 4) TC reagents
 * 5) *Aliquot each at 10-20% excess
 * 6) *PBS (12 mL per group)
 * 7) *autoclaved gelatin (6 mL per group)
 * 8) *Trypsin (1 mL per group)
 * 9) *J1 medium(three tubes per group with precisely 10 mL each)
 * 10) *12 six-well dishes
 * 11) *6 canisters autoclaved Pasteur pipets
 * 12) *6 charged Pipet-Aids

For Next Time

24 hours before Day 7 need to plate 24 well dishes (1 per pair) in DMEM Pre-Txn Media

''From confluent 100mm dish, wash with PBS, add 2 mL trypsin in hood, aspirate incubate(dry) for 10', triterate with 5mL Pre-txn media. Add 0.7mL of this suspension to 14mL pre-txn media (~1:20), place 0.5 ml/well of pre-gelatinized 24 well dish overnight''

Day 7
Long-term preparation:


 * Per group, one 24-well plate with 16 seeded wells.
 * Seed with 105 cells 24 hrs prior to use.
 * Need plasmid with EGFP truncated at 3' end.

Materials required:


 * 1) Lipofectamine (prep 50 &mu;L aliquots)
 * 2) OptiMEM (prep 2 mL aliquots)
 * 3) TC reagents
 * 4) *PBS (10 mL per group)
 * 5) *T'xn Medium (10 mL per group)
 * 6) Sterile eppendorf tubes

Instructors Bench
 * Plasmids for Lipfoection
 * pCX-EGFP
 * pCX-del5
 * pCX-del3


 * Also need
 * pCX-del3 cut with PmeI (blunt)
 * pCX-del3 cut with BamHI (4 bp)
 * pCX-del3 cut with N.BbvcIB (14bp)

In pre-run about 2ug of pCX-del3 was cut for each and recovered 30ul of 0.05ug/ul cut DNA after gel purification
 * (5ul in 500 to measure conc. IOD260 = 50ug/ml

It will be easiest to dilute all DNAs to approx same concentration (pCX-EGFP and pCX-del5 to 0.25ug/ul and all pCX-del3s to 0.05ug/ul would be ideal). Be sure to use STERILE WATER and TC pipets... in Spring of 04 there was terrible contamination of these transfections.

Prep Work For today's lab need 24 well dish (1 per pair) in media WITHOUT antibiotics

Tomorrow will need to remove media and add 1mL fresh Complete DMEM

Confirm FACS time for Day 8 (3 horus starting at 1:30pm)

Day of Lab:


 * Quiz (prepared by TA).

Day 8
FACS time: 1:30-4:30pm

The day will be a repetitive one at the FACS facility. Each group with come with 16 samples to collect. Print out two copies of data for each group, one for them and one for the lab's notes. MES cell culture is done after today!! w00t

Materials required:


 * 1) TC reagents
 * 2) *PBS (20 mL per group)
 * 3) *Trypsin (4 mL per group)
 * 4) *OptiMEM (2 mL per group)
 * 5) FACS tubes
 * 6) *16 per group, plus extras
 * 7) *BD Falcon tubes only, VWR cat # 60819-310 (1000x) or 60819-295 (500x)

Day of Lab:


 * No quiz.
 * TA will run flow cytometer, and faculty will guide student preparations in the lab, or vice-versa.

Agarose Gel

 * 1) DNA gel: 1% agarose gel in 1X TAE, 1 g agarose, 100mL 1X TAE, 2 μL EtBr (wear nitrile gloves when handling EtBr!)
 * 2) *0.5X TBE insteadof 1X TAE for this lab... ?
 * 3) Loading dye for agarose gel: 250 μL 1% XC (xylene cyanol), 750 μL 40% glycerol, 10 μL RNase. Store at RT.
 * 4) 1kb marker: 10 μL 1kb marker stock (in -20 &deg;C freezer), 10 μL loading dye, 90 μL H20

Bacterial growth media

 * LB: 10 g Tryptone, 5 g Yeast Extract, 10 g NaCl per liter. 20g of Agar for plates. Autoclave 30 minutes with stirbar. Pour when ~55°. Let plates dry ON on bench and store in sleeves in 4°. For LB-Amp plates, add the Amp after autoclaving, once the mixture has cooled down.
 * 1) Amp: 100 mg/ml in H20. Filter and store at 4°. Use at 1:1000 in liq media. 2ml/L in plates
 * 2) Top Agar: 10 g Tryptone, 5 g Yeast Extract, 10 g NaCl, 1 g MgCl2*6H20 7 g Agar per liter. Autoclave then aliquot to 50 ml conical tubes or bottles. Store at RT. Melt in microwave in beaker of water, 2’ then keep molten in 55° water bath.

DNA Miniprep

 * 1) Soln I for miniprep: 2.3 ml 40% glucose, 2.5 ml 1M Tris 8, 2 ml 0.5M EDTA. To 100 ml with good H20. Store at RT
 * 2) Soln II for miniprep: equal parts 2% SDS (2g/100 ml H20): 0.4M NaOH (1.6g/100 ml H20). Store components at RT. Mix just enough just before using.
 * 3) Soln III for miniprep: 29.4 g KAc dissolved in 60 ml H20. Add 11.5 ml glacial acetic acid. Bring to 100 ml final volume. Store at RT.

Mammalian cell culture

 * JI Growth Media: 500 ml DMEM (high glucose), 50 ml FBS (Atlanta Biologic, Inc.), 5 ml P/S/G, 1 ml BME, 5 ml NEAA. Filter then add 50 ul LIF. Store 4°C
 * Pre-Transformation Media: 500 ml DMEM (high glucose), 50 ml FBS (Atlanta Biologic, Inc.), 5 ml 100XG, 1 ml BME, 5 ml NEAA. Filter then add 50 ul LIF.
 * J1 culture medium
 * 100 U/ml Pen/Strep
 * 0.3 mg/ml glutamine
 * 0.1 mM BME,
 * 1 mM Nonessential amino acids
 * 10% serum
 * LIF
 * Sterile filter final mixture (0.2 mu;m filter)
 * Gelatin
 * 0.1% TC-grade gelatin prepared in H2O
 * Autoclave mixture and allow to cool before use