20.109(F13): TA notes for module 1

DNA Engineering Module

Current: 20.109(F13)
20.109(F12)
20.109(F11)
20.109(F10)
20.109(F09)(archive)
20.109(F08) (archive)
BE.109(S06) (archive)
20.109(S08) (archive)
Notebook grading checklist

General notes

Enrollment:

• Likely to have at least 2x 8 groups so assume 10 groups/day in terms of materials, except where these additional reagents are difficult to arrange (e.g. extra gels aren't needed, but extra tubes of LB are).

Before the term:

• Pre-run entire cloning procedure (Day 1-4) and save materials in case something goes wrong in the student's experiments
• Streak out pCX-NNX in XL1-blue (NB137) on LB + Amp
• Make a plan for preparing the high volume TC work we do.
• Check materials for TC and verify culture facility ready to run (sterile pipets, traps all working, incubators clean and running)
• Check all links working in student's protocols

Plasmids used: Stocks were prep'd by BioPioneer for F08, F09, F10, F11, F12 version of class.

• pCX-EGFP: stock concentration = 0.5 ug/ul
  • from strain NB124
  • contains full-length EGFP gene
  • Amp resistant
  • f1 ori
  • SV40 ori
  • For PCR (Day 1), dilute stock to about 100 ng/ul (20 ul plasmid, 80 ul H2O)
  • For Transformation (Day 4), dilute stock to about 50 ng/ul (10 ul plasmid, 90 ul H20)

• pCX-NNX: stock concentration = assume 0.5 ug/ul
  • from strain NB137
  • 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: assume 0.5 ug/ul
  • from strain NB168
  • make some stocks of different concentrations for lipofections
  • Alternative experiment (not run in 2012): cut some with PmeI and BamHIfor student's experiment on Day 6 (see day 6 for instructions).

• pCX-EGFP with 5' deletion assume 0.5 ug/ul
  • from strain NB153

Daily Notes

Day 1

Lab has 2 parts:

• Primer design
• PCR

Lab also needs you to:

• Upload key to M1D1 optional assignment
• Check with M1D2 quiz writer

Materials required:

1. PCR Master Mix (2.5X), ~ 50 μL per group
2. DI water, prep 100 μL aliquot per group
3. 5 μL aliquots each of pCX-EGFP (100 ng/ul, 1:5 of stock), D32N-fwd, D32N-rev
4. 2 PCR tubes per group

Day of Lab:

Instructor's Bench: (not needed until near the end of lab)

• Ice bucket
• PCR Master Mix (thawed on ice only just before first group needs it at end of lab)
• Sterile H20, 0.1 ml aliquot/group
• Primers for PCR= D32N-fwd: NO234, D32N-rev: NO235
  • stocks = 100 pmol/ul
• Template for PCR= pCX-EGFP 100 ng/ul
  • pCX-EGFP has been prepared from NB124. For PCR, dilute stock to about 100 ng/ul (1:5 dilution of stock) in sterile H2O.
• Beaker with PCR tubes
• PCR chill racks from -20° (1 per group), taken from freezer as needed

PCR: Check the PCR machine has proper protocol under NK1:

• Step 1: 94C 4min
• Step 2: 94C 1 min
• Step 3: 55C 1 min
• Step 4: 72C 1 min
  • Repeat steps 2-4 34 more times
• Step 5: 72C 10min
• Step 6: 4C hold forever
  • Remember to freeze PCR products when they are ready.

Day 2

Lab has 2 parts:

• Qiagen clean PCR
• Digest bkb, insert
• PCR

Lab also needs you to:

• Upload key to M1D2 optional assignment
• Check with M1D3 quiz writer

Materials required:

1. Qiagen QIAquick PCR purification kit
   • from VWR # 28104, 50 rxns
   • 1 rxn per group
2. pCX-NNX, 20 μL of 0.5 ug/ul stock per group
3. NEB2 Buffer (~10 μL used per group, one 20 μL aliquot per group)
4. EcoRI and XbaI enzymes (~2 μL used per group, aliquot at least 2 x 20ul of each enzyme for each day of lab)
5. sterile DI water (one 100 μL aliquot per group)

Day of Lab:

• 1 ice bucket with ice for each group at their benches
• Distribute and grade student-written quiz
• Thaw and leave aliquots on instructor's bench:
  • PCR products (can leave at RT)
  • pCX-NNX aliquot (20 ul/group, can leave at RT)
  • NEB2 Buffer aliquot (20 ul/group, can leave at RT)
  • EcoRI and XbaI aliquots (20 ul aliquots can be left in cold tray just before students need to use them)

Looking ahead at TC
Check stocks for TC media

• Filtered DMEM Complete
  • 500mL DMEM (high glucose)
  • 50mL serum (FBS from Atlanta Biological)
  • 5mL P/S/G
  • 1mL BME
  • 5mL NEAA
  • LIF
• Filtered DMEM Pre-Txn Media
  • 500mL DMEM (highglucose)
  • 50mL serum (FBS)
  • 5mL 100XS glutamine
  • 1mL BME
  • 5mL NEAA
• Lipofectamine?
• OptiMEM?
• FACS tubes? FACS time?
• Gelatin?

Day 3

This lab has 3 parts:

• Agarose gel electrophoresis
• Qiagen clean up of bkb, frag
• Writing faculty visit

Lab also needs you to:

• Upload key to M1D3 optional assignment
• Check with M1D4 quiz writer
• Collect yellow lab notebook sheets at end of lab to grade and return M1D4

Materials required:

1. Qiagen QIAquick gel extraction kit
   • 2 extractions per group
   • kit from VWR # 28704, 50 rxns
2. isopropanol (one 500 μL aliquot/group)
3. sterile water (one 500 μL aliquot/group)
4. loading dye for agarose gel electrophoresis (one 35 μL aliquot/group)
5. 1% agarose gels prepared in 1X TAE buffer
   • each group has 10 samples, so requires 10 wells
   • prepare 1 gel per two groups, but with 2 combs
   • also prepare 1 more gel (2 combs!), for running purified products
   • Groups will also run the original stock of pCX-NNX (make mix for all to use so each group loads 10 ul (= 5 μL of the stock, 5 μL water and 2 μL loading buffer for each group)
   • Groups will also run 1KB ladder, need 20 ul per group (make at least 3 aliquots)
6. Confirm digital camera OK, + enough paper in thermal printer

Day of Lab:

• Distribute and grade student-written quiz
• Thaw and leave aliquots on instructor's bench:
  • isopropanol
  • sterile water
  • loading dye
  • uncut pCX-NNX to run on gel
  • 1KB ladder alioquots
• After lab, run purified products (2 per group) on an agarose gel at the end of the day - post photograph.
• Freeze DNA at end of day.

Looking ahead at TC

• Cells should be started up
  • Will need one 60mm dish of MES per person on Day 5
  • Will need one 24 well dish of MES per group on Day 6

Day 4

Lab has 3 parts:

• Ligations
• Transformations
• EHS visit

Lab also needs you to:

• Upload key to M1D4 optional assignment
• Check with M1D5 quiz writer
• Return graded notebook pages

Materials required:

1. Buy fresh ligation buffer, T4 ligase from NEB
2. 3 M sodium acetate (one 100 μL aliquot/group)
3. Yeast tRNA (one 25 μL aliquot/group)
4. Cold 100% ethanol (one 1 mL aliquot/group)
5. Cold 70% ethanol (one 3 mL aliquots/group)
6. Sterile DI water (one 100 μL aliquot/group)
7. pCX-EGFP transformation control (dilute stock to 50 ng/ul and aliquot so at least 1 μL is available per group)
8. competent cells
   • XL1-Blue from Agilent, cat # ?200249?
   • 1 tube per group
9. LB+Amp plates, 5 per group + spares
   • may get ~ 40 plates/Liter LB
10. LB liquid medium, stored at RT
11. Amp stock, stored at 4°
12. Check spreaders, refill EtOH beakers and alcohol burners

Day of Lab:

• Distribute and grade student-written quiz
• Set up instructor's bench with all materials needed first for miniprep, then later for transformation.
• Also see "student benchtop" "instructors bench" lists, below

Students Benchtop

• • LB (~5 ml in 15mL conical tube/group)
  • LB+Amp plates (5 per group, so at least 40 per lab)
  • 0.1 mL 3M NaAc (RT)
  • Ice buckets with ice
  • 25 ul tRNA in ice
  • 1 ml 100% EtOH in ice
  • 3 ml 70% EtOH in ice

Instructors Benchtop

• • Ice bucket--these materials should be thawed just before needed by students and kept on ice
    • Ligase and ligation buffer
    • Supercomp cells (need ~200 ul/group, so one tube of cells needed per group)
    • pCX-EGFP transformation control (50 ng/ul)
  • Spreaders, EtOH beakers and alcohol burners (these may need to be refilled with EtOH)

Post Lab Prep

• The night before Day 5: pick colonies for overnight cultures (3 candidates per group, plus 1 pCX-NNX) into 2.5 ml LB+Amp. Grow 37° on roller wheel.

Looking ahead at TC
Next time: TC will need one 60mm dish of confluent J1s PER STUDENT (not per group!) plus two extras for demo.

Day 5

This lab has 2 parts:

• Checking clones on gel
• Practice TC
  

This lab can be chaotic since essentially two labs are running at once: half the students will be in the TC facility splitting cells and the other half will be in the main lab minipreping and digesting their candidates. Halfway through they will switch.
Lab also needs you to:

• Upload key to M1D5 optional assignment
• Check with M1D6 quiz writer, though M1D6 also has lab certification so maybe no quiz also...

Materials required:

Main Lab

1. Miniprep solutions
2. Restriction enzymes and buffers
3. 37° incubator

Student Benchtop

• Liquid cultures (3 candidates, 1 pCX-NNX control per group).
• Miniprep solutions
  • Soln I (one 500 μL aliquot/group)
  • 2% SDS (one 600 μL aliquot/group)
  • 0.4M NaOH (one 600 μL aliquot/group)
  • Soln III (one 800 μL aliquot/group)
  • 100 % ethanol (one 5 mL aliquot/group)
  • 70 % ethanol (one 3 mL aliquot/group)
  • Sterile DI water (one 250 μL aliquot/group)

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

Post Lab Prep

• Agarose gels, 1XTAE, 15 well combs, 2 combs/gel. Two groups can share one gel.

TC

1. TC reagents
   • One 60 mm dish of MES cells near confluence per student
   • Aliquot each at 10-20% excess
   • PBS (12 mL per group)
   • autoclaved gelatin (6 mL per group)
   • Trypsin (1 mL per group)
   • J1 medium(three tubes per group with precisely 10 mL each)
   • 12 six-well dishes
2. Canisters autoclaved Pasteur pipets
3. Charged Pipet-Aids

Looking ahead at TC
24 hours before Day 6 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 6

This lab can be chaotic since essentially two labs are running at once: half the students will be in the TC facility lipofecting cells and the other half will be in the main lab taking a lab certification and doing their defense of their virtual lab work with you. Halfway through lab they will switch. There will also be a student-written quiz to distribute before folks get going (maybe...depending on enrollments).
Other notes on lab:

• There is no M1D6 optional assignment to upload
• You should check with M1D7 quiz writer

Main lab:

see lab certification for details

TC:

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 available for next times

advanced preparation:

• Per group, one 24-well plate with at least 16 seeded wells.
  • Seed with 10⁵ cells 24 hrs prior to use.
• In 2011 this was variable but in 2012 we would like to ask the students to vary the concentration of the two plasmids: If that changes and we need plasmid with EGFP truncated at 3' end, cut with PmeI and a second sample cut with BamHI, then here are the instructions:
  • in 2010 cut this way: 10 ul of delta3 DNA (stock is 0.5 ug/ul) + 80 ul of water + 10 ul 10XNEB4 + 2 ul PmeI or BamHI at 37°C for 30 minutes. This gives a "cut stock" of 0.05 ug/ul and can use 4 ul/transfection if trying for 0.2 ug/transfection.

TC Materials required:

1. Lipofectamine (prep 2.5 μL x [# wells to be transfected + 1] aliquots)
2. OptiMEM (prep 2 mL aliquots)
3. TC reagents
   • PBS (10 mL per group)
   • T'xn Medium (10 mL per group)
4. Sterile eppendorf tubes

TC Instructors Bench

• Plasmids for Lipfoection

• pCX-EGFP (stock is 0.5 ug/ul, so dilute to 0.05 ug/ul in sterile H₂O. Each group will need at least 4 ul of dilution so aliquot 10 ul per group)
• pCX-del5 (stock is 0.5 ug/ul, so dilute to 0.05 ug/ul in sterile H₂O. Each group will need at least 12 ul of dilution so aliquot 20 ul per group)
• pCX-del3 (stock is 0.5 ug/ul, so dilute to 0.05 ug/ul, 0.1 ug/ul and undiluted = 0.5 ug/ul in sterile H₂O. Each group will need between 4 and 8 ul of dilution so aliquot 15 ul per group)

• Also have needed (won't in F12)

Variations of concentration of pCX-del3 (uncut). Alternatively, students have, in the past, tried cuts to plasmid.

• pCX-del3 cut with PmeI (blunt) -- see note above about how to cut DNA
• pCX-del3 cut with BamHI (4 bp) -- see note above about how to cut DNA
• pCX-del3 cut with N.BbvcIB (14bp)-- not offered in 2010

It will be easiest to dilute all DNAs to approx same concentration (~0.05ug/ul would be ideal so students can pipet 4 ul of each for each transfection). Be sure to use STERILE WATER and TC pipets... in Spring of 04 there was terrible contamination of these transfections.

Day of Lab:

• Distribute and grade student-written quiz

Day 7

This day will shuttle the students between TC and the FACS facility in the Koch. FACS time: at least 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
   • PBS (20 mL per group)
   • Trypsin (4 mL per group)
   • OptiMEM (2 mL per group)
2. FACS tubes
   • 16 per group, plus extras
   • BD Falcon tubes only, VWR cat # 60819-310 (1000x) or 60819-295 (500x)

Day of Lab:

• Instructor will distribute and grade student-written quiz
• TA will run flow cytometer, and faculty will guide student preparations in the lab, or vice-versa.

Recipes/Reagents

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. Loading dye for agarose gel: 250 μL 1% XC (xylene cyanol), 750 μL 40% glycerol, 10 μL RNase. Store at RT.
3. 1kb marker: 10 μL 1kb marker stock (in -20 °C freezer), 10 μL loading dye, 90 μL H20

Bacterial growth media

1. 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°.
2. For LB-Amp plates, add Amp (2 ml / L) after autoclaving, once the mixture has cooled down to warm but not hot.
3. Amp: 100 mg/ml in H20. Filter and store at 4°. Use at 1:1000 in liq media. 2ml/L in plates

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