Difference between revisions of "20.109(F15): TA notes for module 1"
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[[20.109(F09)| 20.109(F09)]](archive) <br> | [[20.109(F09)| 20.109(F09)]](archive) <br> | ||
[[20.109(F08)| 20.109(F08)]] (archive)<br> | [[20.109(F08)| 20.109(F08)]] (archive)<br> | ||
− | [ | + | [http://openwetware.org/wiki/BE.109:DNA_engineering | BE.109(S06)] (archive)<br> |
[[20.109(S08):Module 1| 20.109(S08)]] (archive)<br> | [[20.109(S08):Module 1| 20.109(S08)]] (archive)<br> | ||
[[Media:TA NotebookGrading.jpg| Notebook grading checklist]] | [[Media:TA NotebookGrading.jpg| Notebook grading checklist]] | ||
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'''''Looking ahead at TC'''''<br> | '''''Looking ahead at TC'''''<br> | ||
Check stocks for TC media | Check stocks for TC media | ||
− | * | + | *J1 media (see Recipes/Reagents section below) |
− | + | *J1 media without P/S (see Recipes/Reagents section below) | |
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*Lipofectamine? | *Lipofectamine? | ||
*OptiMEM? | *OptiMEM? | ||
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*Agarose gel electrophoresis | *Agarose gel electrophoresis | ||
*Qiagen clean up of bkb, frag | *Qiagen clean up of bkb, frag | ||
− | * | + | *How to pour an agarose gel |
Lab also needs you to: | Lab also needs you to: | ||
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#* also prepare 1 more gel (2 combs!), for running purified products | #* 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 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 kb ladder, need 20 μL per group (make at least 3 aliquots) | + | #* Groups will also run 1 kb ladder, need 20 μL per group (make at least 3 aliquots) |
#Confirm digital camera OK, + enough paper in thermal printer | #Confirm digital camera OK, + enough paper in thermal printer | ||
'''Day of lab:''' | '''Day of lab:''' | ||
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*Thaw and leave aliquots on instructor's bench: | *Thaw and leave aliquots on instructor's bench: | ||
**isopropanol | **isopropanol | ||
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**loading dye | **loading dye | ||
**uncut pCX-NNX to run on gel | **uncut pCX-NNX to run on gel | ||
− | ** | + | **1 kb ladder aliquots |
*After lab, run purified products (2 per group) on an agarose gel at the end of the day - post photograph. | *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. | *Freeze DNA at end of day. | ||
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*Ligations | *Ligations | ||
*Transformations | *Transformations | ||
− | * | + | *BE Communication Lab visit (on abstracts) |
Lab also needs you to: | Lab also needs you to: | ||
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# Buy fresh ligation buffer, T4 ligase from NEB | # Buy fresh ligation buffer, T4 ligase from NEB | ||
# 3 M sodium acetate (one 100 μL aliquot/group) | # 3 M sodium acetate (one 100 μL aliquot/group) | ||
− | # Yeast tRNA (one 25 μL aliquot/group) | + | # Yeast tRNA 12.5mg/ml (one 25 μL aliquot/group) |
# Cold 100% ethanol (one 1 mL aliquot/group) | # Cold 100% ethanol (one 1 mL aliquot/group) | ||
# Cold 70% ethanol (one 3 mL aliquots/group) | # Cold 70% ethanol (one 3 mL aliquots/group) | ||
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*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 °C on roller wheel. | *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 °C on roller wheel. | ||
'''''Looking ahead at TC'''''<br> | '''''Looking ahead at TC'''''<br> | ||
− | Next time: TC will need one 60 mm dish of confluent J1s '''PER STUDENT''' (not per group!) plus two extras for demo. | + | Next time: TC will need one 60 mm dish of confluent J1s '''PER STUDENT''' (not per group!) plus two extras for demo. Plating 500,000 cells per 60-mm dish ensures confluence on the next day. |
===[[20.109(F15):Examine candidate clones & tissue culture (Day5)| Day 5]]=== | ===[[20.109(F15):Examine candidate clones & tissue culture (Day5)| Day 5]]=== | ||
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#*PBS (12 mL per group) | #*PBS (12 mL per group) | ||
#*'''autoclaved''' gelatin (6 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) | #*J1 medium (three tubes per group with precisely 10 mL each) | ||
#*12 six-well dishes | #*12 six-well dishes | ||
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'''''Looking ahead at TC'''''<br> | '''''Looking ahead at TC'''''<br> | ||
− | 24 hours before Day 6 need to plate 24 well dishes (1 per pair) in | + | 24 hours before Day 6 need to plate 24-well dishes (1 per pair) in J1 ''without antibiotics''<br> |
− | + | Pre-gelatinize 24-well dishes with 0.1% gelatin (in DPBS) for 5 minutes. Aspirate gelatin and add 0.5 mL J1 media without P/S to each of the 17 wells. After trypsinizing (T75 flask of) J1 cells, add 150,000 cells per well. | |
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===[[20.109(F15):Lipofection (Day6) | Day 6]]=== | ===[[20.109(F15):Lipofection (Day6) | 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 | + | 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. <br> |
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===='''TC:'''==== | ===='''TC:'''==== | ||
− | For today's lab need 24 well | + | For today's lab need 24-well dishes (1 per pair) in media WITHOUT antibiotics.<br> |
− | Tomorrow will need to remove media and add | + | Tomorrow (if 48-hour lipofection treatment) will need to remove media and add 0.5 mL fresh complete J1 media.<br> |
Confirm FACS available for next times<br> | Confirm FACS available for next times<br> | ||
'''Advanced preparation:''' | '''Advanced preparation:''' | ||
− | * Per group, one 24-well plate with | + | * Per group, one 24-well plate with 17 seeded wells. |
− | ** Seed with 10<sup>5</sup> cells 24 | + | ** Seed with 1.5 * 10<sup>5</sup> cells 24 h 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 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 μL of Δ3 DNA (stock is 0.5 μg/μL) + 80 μL of water + 10 μL 10x NEB4 + 2 μL ''PmeI'' or ''BamHI'' at 37 °C for 30 minutes. This gives a "cut stock" of 0.05 μg/μL and can use 4 μL/transfection if trying for 0.2 μg/transfection. | **in 2010 cut this way: 10 μL of Δ3 DNA (stock is 0.5 μg/μL) + 80 μL of water + 10 μL 10x NEB4 + 2 μL ''PmeI'' or ''BamHI'' at 37 °C for 30 minutes. This gives a "cut stock" of 0.05 μg/μL and can use 4 μL/transfection if trying for 0.2 μg/transfection. | ||
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#TC reagents | #TC reagents | ||
#*PBS (10 mL per group) | #*PBS (10 mL per group) | ||
− | #* | + | #*J1 media without P/S (10 mL per group) |
#Sterile eppendorf tubes | #Sterile eppendorf tubes | ||
'''TC Instructors Bench''' | '''TC Instructors Bench''' | ||
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:*pCX-del3 cut with ''BamHI'' (4 bp) -- 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 | :*pCX-del3 cut with ''N.BbvcIB'' (14bp)-- not offered in 2010 | ||
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It will be easiest to dilute all DNAs to approx same concentration (~0.05 μg/μL would be ideal so students can pipet 4 μL 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. | It will be easiest to dilute all DNAs to approx same concentration (~0.05 μg/μL would be ideal so students can pipet 4 μL 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. | ||
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===[[20.109(F15):FACS analysis (Day7)| Day 7]]=== | ===[[20.109(F15):FACS analysis (Day7)| Day 7]]=== | ||
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===Agarose gel=== | ===Agarose gel=== | ||
− | # DNA gel: 1% agarose gel in 1X TAE, 1 g agarose, 100mL 1X TAE, 10 | + | # DNA gel: 1% agarose gel in 1X TAE, 1 g agarose, 100mL 1X TAE, 10 |