McGill/Team 1: Fluorescence Complementation

From 2007.igem.org

(Difference between revisions)
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#Place into 37°C incubator at 3:55PM
#Place into 37°C incubator at 3:55PM
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'''Next''': miniprep and screen the DNA.
+
'''Next''': seed, miniprep and screen the DNA.
=== June 21 ===
=== June 21 ===
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=== June 22 ===
=== June 22 ===
 +
'''Today''': miniprep DNA from July 20th transformation
 +
 +
I - Miniprep of Colonies from R25, R400, C25, C300 (indication the biobrick and the amount in uL that was plated)
 +
#Transfer 1.5mL of cells suspended in LB into a microcentrifuge tube.
 +
#Centrifuge for 2 min at max speed (14000 rpm)
 +
#Remove supernatant with pipette and discard.
 +
#Add 250uL of buffer P1 and 250uL of buffer P2
 +
#Close and invert tubes 6 times
 +
#Add 350 uL of buffer N3 (''within 5 min'' of adding buffer P2)
 +
#Close and invert 6 times
 +
#Centrifuge for 10 min at 13000 rpm
 +
#Carefully pipette off the supernatant into a Qiaprep spin column. ''NB: for sample R400 a large amount of supernatant was discarded.  No reason, stupid move by Tim.''
 +
#Centrifuge spin column for 60 sec
 +
#Discard flow-through
 +
#Add 750uL of buffer PE
 +
#Centifuge for 60 sec (max speed)
 +
#Add 50uL of sterile water (of EB buffer could be used)
 +
#Let stand for 1 min then centrifuge for 1 min
 +
#Transfer flow-through to a new micro-centrifuge tube (one with a cap) with a pipette.
 +
#Stored in -20°C freezer (green box labelled "Team 2")
 +
#Remaining cells in LB stored in small fridge
 +
 +
'''Next''': Screen the DNA.

Revision as of 13:13, 6 September 2007

McGill Home

Go to Team 2

May 2007
Su M Tu W Th F Sa
1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30 31
June 2007
Su M Tu W Th F Sa
      1 2
3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30



Contents

May 2007

May 14

  1. Made the same as May 15, but did not use Type 1 (milliQ) H2O, so the solutions were discarded.

May 15

  1. Made 1M CaCl2, and glycerol/CaCl2 solutions
    1. 1M CaCl2 Solution: 100mL was made
      1. CaCl2 Dihydrate = 146.986 g/mol
      2. 100mL of 1M CaCl2 requires 14.699g CaCl2 Dihydrate
    2. Glycerol/CaCl2 Solution: 30mL was made
      1. 5.00mL of 60% glycerol solution
      2. 3.00mL of 1M CaCl2
      3. 22.00mL of Type 1 (milliQ) H2O

May 16

  1. Transformation of Cells:
    1. Transformed 1 vial of cells with Jun vector, and another vial with Fos vector (1uL of vector each); Sterile
    2. Used 400uL of LB (sterile) for each vial
    3. Incubated for 1 hour in shaker at 37C, then incubate overnight at 37C

May 30

  1. Test PCR Machine


June 2007

June 4

  1. Made Kanamycin plates
    1. 200mL of Agar
    2. 200mL of 2x LB
    3. 2mL of Kanamycin

June 20

Today: extract DNA from plates, transform, plate on amp plates.

I - Extraction of DNA from Plate (5M and 9G)

  1. Puncture foil with pippette tip
  2. Add 15uL of Type 1 water
  3. Remove all liquid (with DNA in solution)

II - Transformation of extracted DNA

  1. Chill cells on ice for 10 min
  2. Heat shock cells at 42°C for 30 sec
  3. Add DNA for cells
  4. Add 500uL of 42°C SOC medium to each vial
  5. Incubate on ice for 1 min
  6. Place in 37°C shaker incubator for 1h 30min
  7. Plate onto 4 separate amp plates
    1. 25uL R0062
    2. 25uL C0060
    3. 400uL R0062
    4. 300uL C0060
  8. Place into 37°C incubator at 3:55PM

Next: seed, miniprep and screen the DNA.

June 21

Checked colonies in the 37°C incubator. Well formed colonies were on each plate, so plates (all 4) were transferred to the small fridge (middle shelf on the right).

June 22

Today: miniprep DNA from July 20th transformation

I - Miniprep of Colonies from R25, R400, C25, C300 (indication the biobrick and the amount in uL that was plated)

  1. Transfer 1.5mL of cells suspended in LB into a microcentrifuge tube.
  2. Centrifuge for 2 min at max speed (14000 rpm)
  3. Remove supernatant with pipette and discard.
  4. Add 250uL of buffer P1 and 250uL of buffer P2
  5. Close and invert tubes 6 times
  6. Add 350 uL of buffer N3 (within 5 min of adding buffer P2)
  7. Close and invert 6 times
  8. Centrifuge for 10 min at 13000 rpm
  9. Carefully pipette off the supernatant into a Qiaprep spin column. NB: for sample R400 a large amount of supernatant was discarded. No reason, stupid move by Tim.
  10. Centrifuge spin column for 60 sec
  11. Discard flow-through
  12. Add 750uL of buffer PE
  13. Centifuge for 60 sec (max speed)
  14. Add 50uL of sterile water (of EB buffer could be used)
  15. Let stand for 1 min then centrifuge for 1 min
  16. Transfer flow-through to a new micro-centrifuge tube (one with a cap) with a pipette.
  17. Stored in -20°C freezer (green box labelled "Team 2")
  18. Remaining cells in LB stored in small fridge

Next: Screen the DNA.

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