Difference between revisions of "Team:Stony Brook/Notebook/Cancer-W14"

 
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Latest revision as of 07:54, 19 October 2016

Week 14: 9/26 - 10/2

Week 14: 9/26 - 10/2



9/29

Toggle 2 Gel Construct using Phire Polymerase



PCR Ran of Toggle 2:
    Phase Temperature (°C) Time (sec)
    Initial Denaturation 98 60s
    Denaturation 98 20s
    Annealing Ranged from 72-60 degrees for each tube. Did a different annealing temperature for each PCR. 10s
    Extension 72 #
    Final Extension 72 #
    Ran 32 cycles


9/30

  • Ran PCR of CRISBu
  • Ligation of CrisBu into pEP352GAP
  • Nanodrop and PCR of Toggle 2
PCR Toggle 2:
    Contents ul
    Phire 10
    H2O 7, 6, and 4 for three tubes respectively
    10 uM F Primer 0.5, 1.0, and 2.0 for three tubes respectively
    10 uM R Primer 0.5, 1.0, and 2.0 for three tubes respectively
    100ug/ul Construct 2 uL

PCR Settings Toggle 2:

    Phase Temperature (°C) Time (sec)
    Initial Denaturation 98 60s
    Denaturation 98 10s
    Annealing 69 10s
    Extension 72 40s
    Final Extension 72 60s


10/1

Constructs of T1 and T2

  • Ran a gel of the constructs
  • Set up tubes for TSS Competent cells
CRISBU
Content Experimental ul
Cutsmart 5
EcoRI-HF 1
XhoI 1
DNA 43
Toggle 1
Content Experimental ul
Cutsmart 5
EcoRI-HF 1
SpeI-HF 1
DNA 28
  • Filled to 50ul with water
Toggle 2
Content Experimental ul
Cutsmart 5
EcoRI-HF 1
PstI-HF 1
DNA 35
  • Filled to 50ul with water

Ligations

pEPGAP
Content ul
10X T4 Buffer 2
Vector 2
Insert 0
H2O 15
Ligase 1
pEPGAP/CRISBU
Content ul
10X T4 Buffer 2
Vector 2
Insert 2
H2O 13
Ligase 1
pEPGAP-P
Content ul
10X T4 Buffer 2
Vector 4
Insert 0
H2O 13
Ligase 1
pEPGAP-P/CRISBU
Content ul
10X T4 Buffer 2
Vector 4
Insert 4
H2O 8
Ligase 1
pSBIA2/T1/T2
Content ul
10X T4 Buffer 2
Vector 1
Insert 13.52
H2O 2.5
Ligase 1
pSBIA2
Content ul
10X T4 Buffer 2
Vector 1
Insert 0
H2O 16
Ligase 1
pSBIA2-p
Content ul
10X T4 Buffer 2
Vector 2
Insert 0
H2O 15
Ligase 1
pSBIA2-P/T1/T2
Content ul
10X T4 Buffer 2
Vector 2
Insert 13.50
H2O 2.5
Ligase 1


10/2

Transformations

  1. Thaw TSS cells on ice
  2. Add 2ul of prepped plasmid. Pipette to mix
  3. Sit for 30 minutes on ice
  4. Incubate cells for 30s at 43°C
  5. Incubate on ice for 2 minutes
  6. Add 1ml of 50C
  7. Incubate for 30 minutes at 37°C. Shake
  8. Spread 100-300ul on plates
  9. Grow overnight at 37°C
  10. Save the rest as liquid cultures

Plates Used:

  • Plate 1: pEPGAP
  • Plate 2: pEPGAP/CRISBU
  • Plate 3: pEPGAP-P
  • Plate 4:
  • Plate 5: pSBIA2/T1/T2
  • Plate 6: pSBIA2
  • Plate 7: pSBIA2-P
  • Plate 8: