20.109(S14): TA notes for module 2

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(General notes)
(Day 4)
Line 103: Line 103:
'''Materials required:'''  
'''Materials required:'''  
-
Digests and second day of Westerns<br>
+
*Digests
-
<br>
+
**Aliquot exactly 3.5 &mu;g DNA per pair
-
Thaw buffers, make aliquots<br>
+
**Water and NEB buffer aliquots, a few
-
Place appropriate enzymes in orange rack<br>
+
*Purification
-
 
+
**loading dye aliquots
-
Used Qiagen kit:<br>
+
**1% agarose gels
-
Make aliquots of tubes (2, pseudo-sterile), DI water (100uL), QG (550uL), PE (1.8mL), EB (200uL), isopropanol, spin columns<br>
+
**TAE buffer
-
Pour gels<br>
+
**Qiagen aliquots: DI water (100uL), QG (550uL), PE (1.8mL), EB (200uL), isopropanol
-
Make TE buffer<br>
+
**Only put out as many columns as students should need
-
Make enough ladder (1kb)<br>
+
**Have psuedo/once-sterile eppendorf tubes out
-
Prepare control digest, if desired<br>
+
*Western Day 2
-
Set out ice buckets<br>
+
**Plenty of TBS-T, in case folks over-wash
-
<br>
+
***?Dilute 10% tween (10X)
-
<u>Western prep</u>:<br>
+
***?Dilute 10x TBS in total desired volume of water
-
Aliquots of secondary Ab (goat and-rabbit alkaline phosphate conjugate)<br>
+
**Shaker area in fume hood cleaned up
-
Thaw development buffer / reagents A and B<br>
+
**A few aliquots of GAR-AP
-
Make chilled TBS-T (~2L total)<br>
+
**I believe 24 mL distilled H2O aliquots were pre-prepped for them?
-
Recipe:
+
-
Dilute 10% tween (10X)<br>
+
-
Dilute 10x TBS in total desired volume of water<br>
+
-
<br>
+
-
<br>
+
-
'''Day of Lab (R/F):'''<br>
+
-
Set out water bath/heat block (50C)<br>
+
-
Prepare 6xNEB loading dye stations for when students' digests are ready<br>
+
-
Start weighing station for eppendorf tubes 10 minutes before gel is set to finish.<br>
+
 +
'''Day of Lab (R/F):'''
 +
*Set out ice buckets
 +
*Prepare 50 &deg;C heat block
 +
*Place appropriate enzymes in orange freezer rack in alphabetical order
 +
*Put out DNA ladder on cold rack as well
 +
*Encourage students to pre-weight eppendorfs.
 +
*Pipet-aids out for aliquotting 9 mL TBS-T
 +
*Thaw/refrigerate development buffer if not already in fridge
 +
*Briefly thaw reagents A and B and keep in the dark
 +
*Measure 260 and 280 nm values for each pair's DNA
'''After Lab:'''
'''After Lab:'''
 +
 +
*Take and post pictures of blots if students did not
'''How it went:'''
'''How it went:'''
-
Generally smooth, students in small section finished with ~20 min to spare.
+
 
 +
*Generally smooth, students in small section finished with ~20 min to spare.
 +
*Next year, consider consistently running single cut DNA alongside the digests, and even separate out a bit longer to avoid single-cut contamination
===Day 5===
===Day 5===

Revision as of 11:17, 4 June 2014

20.109(S14): Laboratory Fundamentals of Biological Engineering

Home        Schedule Spring 2014        Assignments       
Module 1        Module 2        Module 3              

Contents

General notes

See also Dropbox Excel document for aliquoting amounts.

Scheme: Each pair of students will test K1 and xrs6 cells for Ku80 production. They will then assess the ability of K1, xrs6, and DNA-PKcs inhibited cells to repair DNA damage via plasmid based assay and flow cytometry.

Key preparation:

Cells:

  • Prepare T25 flasks of K1 and xrs6 for every team

Westerns:

  • Prepare transfer buffer

DNA plasmids:

  • Aliquot plasmids

Lipofection: Flow cytometry:


Note to TA:

  • While you read the wiki, test each link, and make any link descriptions bold that are not already.
  • As you read the protocols, add reagent calculations to the Dropbox Excel doc as needed.

Day-by-day

Day 1

Materials required:

1. Aliquots of PBS, CHO media, and 0.25% Phenol Red Trypsin for each group 2. T25 flask of K1 and xrs6 cells for each group

  • Flasks for next day seeded at 600,000 cells
  • Flasks for day after next, seeded at 400,000 cells
  • Flasks for 3 days in future, seeded at 200,000 cells

Day of Lab (T/W):

  • No Quiz
  • Prepare remaining aliquots for students
  • Warm up media, PBS, and trypsin half hour before students use reagents

Additional Prep.:

  • Digest pMAX-BFP-2MCS2 for testing damage topologies
  • Pour gel for purification

After Lab:

  • Aliquot new shipment of trypsin

How it went:

Day went pretty smoothly; not many questions on Part 4 exercise.

Day 2

Materials required: Please read as much as you can of Parts 1-3 and create an automated worksheet (e.g., in Excel) that will perform the required calculations for the day. A partially prepared worksheet is linked here. We will NOT collect your final worksheet this time; it is only for your benefit.

Prep. For westerns: Make aliquots: Chill scrapers RIPA lysis buffer Protease inhibitor (last minute) Eppendorf tubes PBS for wash Ice buckets

Set up boiling station: Turn on hot plates with water in glass bowl with boiling chips Aliquot beta-mercap. Don't forget to show students the cap covers

Set up westerns: Make sds running buffer Make transfer buffer (chilled) Set up gels(remove strips, place in boxes, remove combs, wash wells with running buffer) Night before: freeze ice blocks

Presoak filter paper and scotch pads in transfer buffer for 30 min prior to blot.

Make sure we have blocking buffer.


Day of Lab (R/F):

How it went:

Day 3

Materials required: None, your brains :)

Day of Lab (T/W): First quiz

After Lab:

How it went:

Day 4

Materials required:

  • Digests
    • Aliquot exactly 3.5 μg DNA per pair
    • Water and NEB buffer aliquots, a few
  • Purification
    • loading dye aliquots
    • 1% agarose gels
    • TAE buffer
    • Qiagen aliquots: DI water (100uL), QG (550uL), PE (1.8mL), EB (200uL), isopropanol
    • Only put out as many columns as students should need
    • Have psuedo/once-sterile eppendorf tubes out
  • Western Day 2
    • Plenty of TBS-T, in case folks over-wash
      • ?Dilute 10% tween (10X)
      • ?Dilute 10x TBS in total desired volume of water
    • Shaker area in fume hood cleaned up
    • A few aliquots of GAR-AP
    • I believe 24 mL distilled H2O aliquots were pre-prepped for them?

Day of Lab (R/F):

  • Set out ice buckets
  • Prepare 50 °C heat block
  • Place appropriate enzymes in orange freezer rack in alphabetical order
  • Put out DNA ladder on cold rack as well
  • Encourage students to pre-weight eppendorfs.
  • Pipet-aids out for aliquotting 9 mL TBS-T
  • Thaw/refrigerate development buffer if not already in fridge
  • Briefly thaw reagents A and B and keep in the dark
  • Measure 260 and 280 nm values for each pair's DNA

After Lab:

  • Take and post pictures of blots if students did not

How it went:

  • Generally smooth, students in small section finished with ~20 min to spare.
  • Next year, consider consistently running single cut DNA alongside the digests, and even separate out a bit longer to avoid single-cut contamination

Day 5

Materials required:

Day of Lab (T/W):

After Lab:

How it went:

Day 6

Materials required:

Day of Lab (R/F):

After Lab:

How it went:

Day 7

Materials required:

Day of Lab (T/W):

After Lab:

How it went:

Day 8

Materials required:

Day of Lab (R/F):

After Lab:

How it went:
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