Difference between revisions of "20.109(S08): TA notes for module 1"
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*Remember to freeze PCR products when they are ready. | *Remember to freeze PCR products when they are ready. | ||
− | '''Instructor's Bench''' | + | '''Instructor's Bench:''' |
*Ice bucket | *Ice bucket | ||
*PCR Master Mix (taken out of -20C midway through labtime) | *PCR Master Mix (taken out of -20C midway through labtime) | ||
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*Template for PCR (pCX-EGFP 100 ng/ul) | *Template for PCR (pCX-EGFP 100 ng/ul) | ||
*Beaker with PCR tubes | *Beaker with PCR tubes | ||
+ | |||
'''Prep Work''' | '''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. | 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. | ||
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#*order # 28104, 50 rxns | #*order # 28104, 50 rxns | ||
#*1 rxn per group | #*1 rxn per group | ||
− | #pCX-NNX, | + | #pCX-NNX, 20 μL per group |
#NEB2 Buffer (~5 μL used per group) | #NEB2 Buffer (~5 μL used per group) | ||
#EcoRI and XbaI enzymes (~2 μL used per group) | #EcoRI and XbaI enzymes (~2 μL used per group) | ||
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*Thaw PCR products and template on ice. | *Thaw PCR products and template on ice. | ||
− | + | ||
+ | '''''Prep Work''''' | ||
+ | |||
Photocopy pages 232 to 233 from NEB catalog for students to read for next time. | Photocopy pages 232 to 233 from NEB catalog for students to read for next time. | ||
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#*pCX-NNX, XbaI cut only | #*pCX-NNX, XbaI cut only | ||
#*pCX-NNX, EcoRI cut only | #*pCX-NNX, EcoRI cut only | ||
− | |||
#TC Room | #TC Room | ||
#*Razors, Face Masks, Film/Camera, Transilluminator, Spatulas, Kimwips, Eppendorf, Pen, EtBR waste jar | #*Razors, Face Masks, Film/Camera, Transilluminator, Spatulas, Kimwips, Eppendorf, Pen, EtBR waste jar | ||
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*Run purified products (2 per group) on an agarose gel at the end of the day - post photograph. | *Run purified products (2 per group) on an agarose gel at the end of the day - post photograph. | ||
*Freeze DNA at end of day. | *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 | ||
===[[20.109%28S08%29:DNA_engineering/DNA_ligation_and_bacterial_transformation_%28Day_4%29 | Day 4]]=== | ===[[20.109%28S08%29:DNA_engineering/DNA_ligation_and_bacterial_transformation_%28Day_4%29 | Day 4]]=== | ||
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'''Day of Lab:''' | '''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). | *Quiz (prepared by TA). | ||
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'''Materials required:''' | '''Materials required:''' | ||
+ | '''Student Benchtop''' | ||
*Liquid cultures (3 candidates, 1 pCX-NNX control per group). | *Liquid cultures (3 candidates, 1 pCX-NNX control per group). | ||
*Miniprep solutions | *Miniprep solutions | ||
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**Sterile DI water (prep 250 μL aliquots) | **Sterile DI water (prep 250 μL aliquots) | ||
*Digests: have all 4 NEB buffers and many restriction enzymes available | *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!! | ||
===[[20.109%28S08%29:DNA_engineering/Restriction_map_and_tissue_culture | Day 6]]=== | ===[[20.109%28S08%29:DNA_engineering/Restriction_map_and_tissue_culture | 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:''' | '''Long-term preparation:''' | ||
* Per student, one 60 mm dish of MES cells near confluence | * Per student, one 60 mm dish of MES cells near confluence | ||
+ | * Six 1% agarose gels with EtBr (10 wells/row) | ||
'''Materials required:''' | '''Materials required:''' | ||
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#*Trypsin (1 mL per group) | #*Trypsin (1 mL per group) | ||
#*J1 medium(three tubes per group with precisely 10 mL each) | #*J1 medium(three tubes per group with precisely 10 mL each) | ||
+ | #*12 six-well dishes | ||
+ | #*6 canisters autoclaved Pasteur pipets | ||
+ | #*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'' | ||
===[[20.109%28S08%29:DNA_engineering/Lipofection_%28Day_7%29 | Day 7]]=== | ===[[20.109%28S08%29:DNA_engineering/Lipofection_%28Day_7%29 | Day 7]]=== | ||
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#*PBS (10 mL per group) | #*PBS (10 mL per group) | ||
#*T'xn Medium (10 mL per group) | #*T'xn Medium (10 mL per group) | ||
+ | #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:''' | '''Day of Lab:''' | ||
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===[[20.109%28S08%29:DNA_engineering/FACS_analysis_%28Day_8%29 | Day 8]]=== | ===[[20.109%28S08%29:DNA_engineering/FACS_analysis_%28Day_8%29 | 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:''' | '''Materials required:''' | ||
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#*PBS (20 mL per group) | #*PBS (20 mL per group) | ||
#*Trypsin (4 mL per group) | #*Trypsin (4 mL per group) | ||
− | #*OptiMEM (2 mL per group) | + | #*OptiMEM (2 mL per group) |
#FACS tubes | #FACS tubes | ||
#*16 per group, plus extras | #*16 per group, plus extras | ||
− | #*BD Falcon tubes only, VWR cat # 60819-310 | + | #*BD Falcon tubes only, VWR cat # 60819-310 (1000x) or 60819-295 (500x) |
'''Day of Lab:''' | '''Day of Lab:''' | ||
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===Agarose Gel=== | ===Agarose Gel=== | ||
− | # DNA gel: 1% agarose gel in 1X TAE, 1 g agarose, 100mL 1X TAE, 2 | + | # DNA gel: 1% agarose gel in 1X TAE, 1 g agarose, 100mL 1X TAE, 2 |