Difference between revisions of "20.109(S08): TA notes for module 1"
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** f1 ori | ** f1 ori | ||
** some altered restriction sites compared to pCX-EGFP, and SV40 ori deleted | ** some altered restriction sites compared to pCX-EGFP, and SV40 ori deleted | ||
+ | |||
+ | *pCX-EGFP with 3' deletion - <font color = red>still have this in lab, how labeled?</font color> | ||
==Daily Notes== | ==Daily Notes== | ||
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*Lab orientation quiz (not a TA quiz) will be taken today. | *Lab orientation quiz (not a TA quiz) will be taken today. | ||
*Remember to freeze PCR products when they are ready. | *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 | ||
===[[20.109%28S08%29:DNA_engineering/Clean_and_cut_DNA_%28Day_2%29 | Day 2]]=== | ===[[20.109%28S08%29:DNA_engineering/Clean_and_cut_DNA_%28Day_2%29 | Day 2]]=== | ||
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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) | ||
Line 55: | Line 91: | ||
*Quiz (prepared by TA). | *Quiz (prepared by TA). | ||
*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. | ||
+ | |||
+ | '''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 | ||
+ | ---- | ||
===[[20.109%28S08%29:DNA_engineering/Agarose_gel_electrophoresis_%28Day_3%29 | Day 3]]=== | ===[[20.109%28S08%29:DNA_engineering/Agarose_gel_electrophoresis_%28Day_3%29 | Day 3]]=== | ||
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'''Materials required:''' | '''Materials required:''' | ||
− | #Qiagen QIAquick gel extraction kit | + | #Qiagen QIAquick gel extraction kit |
#*2 extractions per group | #*2 extractions per group | ||
#*order # 28704, 50 rxns | #*order # 28704, 50 rxns | ||
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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 |
+ | #*Razors, Face Masks, Film/Camera, Transilluminator, Spatulas, Kimwips, Eppendorf, Pen, EtBR waste jar | ||
+ | #Minigel (1%) to check recovery of student's fragments after class | ||
'''Day of Lab:''' | '''Day of Lab:''' | ||
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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]]=== | ||
+ | |||
+ | '''Long-term preparation:''' | ||
+ | |||
+ | * Per group, one 24-well plate with 16 seeded wells. | ||
+ | ** Seed with 10<sup>5</sup> cells 24 hrs prior to use. | ||
+ | * Need plasmid with EGFP truncated at 3' end. | ||
+ | |||
+ | '''Materials required:''' | ||
+ | |||
+ | #Lipofectamine (prep 50 μL aliquots) | ||
+ | #OptiMEM (prep 2 mL aliquots) | ||
+ | #TC reagents | ||
+ | #*PBS (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:''' | ||
*Quiz (prepared by TA). | *Quiz (prepared by TA). | ||
+ | |||
+ | ===[[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:''' | ||
+ | |||
+ | #TC reagents | ||
+ | #*PBS (20 mL per group) | ||
+ | #*Trypsin (4 mL per group) | ||
+ | #*OptiMEM (2 mL per group) | ||
+ | #FACS tubes | ||
+ | #*16 per group, plus extras | ||
+ | #*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. | ||
==Recipes/Reagents== | ==Recipes/Reagents== | ||
===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 |