Team:Stony Brook/Notebook/Cancer-W4

Week 4: 7/18-7/22

7/18

Ran a digest of PEP352GAP vector

    Contents Experimental (ul) Control (ul)
    H2O 19.9 20.9
    10x Cutsmart 2.5 2.5
    XnoI .5 X
    PvoII-GF .5 ul X
    DNA (306.6ug/ul) 1.63 1.63
  • Mix tubes by flicking a few times
  • Spin down in centrifuge
  • Reactions stopped by adding 4.16 ul of purple loading dye to tubes

Running Gel

Lane:

  1. Ladder
  2. Vector Digest - Experimental
  3. Vector Digest - Control

After gel run:

  • Excised Vector Digest - Experimental
  • Gel purified → 180mg of gel → 720 ul of dissolving buffer
  • Ran Nanodrop
    Nanodrop Concentration (ug/ul) 260/280
    Vector Digest 20.4 2.47
    Construct Digest 2.3 -7.40
Ran gel on a digestion of vector + construct with ladder

Lane:

  1. Construct Control
  2. Construct Experiment
  3. Ladder
  4. Vector Experiment
  5. Vector Control
  • Excised Lane 2 (Construct Experiment) and put in fridge
"This is why I'm never going to have children" -Ryan


7/19

Digest of Dean Vector (25ul Reaction)
    Contents Experimental (ul) Control (ul)
    H2O 17.80 18.80
    10x Cutsmart 1 1
    XhoI .5 X
    PvoII-HF .5 X
    DNA (96.3ug/ul) 5.19 5.19
  • Incubated at 37°C for 1hr. Purple loading dye, no SDS added (4.16ul)
  • Restreaked Dean vector colony
  • Gel extraction of construct digest excise
  • 200mg of gel → 800ul of dissolving buffer
  • Dissolved at 47°C
  • Also made a new 10X TAE buffer
Ran a Nanodrop
    Contents Concentration (ug/ul)
    YEP352GAP Miniprep - 7/13 31.7
    YEP352GAP Miniprep - 7/12 10.8
    Purification PCR Construct 1 - 7/15 24.0
    Purification of PCR Construct 2 - 7/15 50.4
PCR of CR-1 PCR Product - Main Direct Program 1 (2 replicates - 50ul Reaction):
    Contents Amount (ul)
    H2O 19
    Phire 25
    10uM F Primer 2.5
    10uM R Primer 2.5
    Template 1
Digest of PEP352GAP
    Content Experimental (ul) Control (ul)
    H2O 11.6 12.6
    10X Cutsmart 2.5 2.5
    XhoI .5 X
    PvoII-HF .5 X
    DNA(50.4 ug/ul) 9.92 9.92
Ran a gel

Lane:

  1. Ladder
  2. PCR Tube #1
  3. PCR Tube #2
  4. Digest Vector - Experiment
  5. Digest Vector - Control
  • Gel smeared
Ran a second gel
  • Ran with the same setup as the gel above
  • Only PCR Tube #1 worked
  • Gel purified and PCR purified
Ran a Nanodrop
    Contents Concentration (ug/ul)
    PCR Purified - 7/19 95.1
    Gel Purified PCR Product - 7/19 5.7


7/20

Q5 Master Mix (2X) PCR of construct - 2 Reactions (25ul)
    Content Rx #1 (ul) Rx #2 (ul)
    Q5 Master Mix 12.5 12.5
    10mM F Primer 1.25 1.25
    10mM R Primer 1.25 1.25
    Template (95.1ug/ul) .5 1
    Milli-Q H2O 9.5 9.0
PCR Setup
  • Ran for 35 cycles
  • Lid heated to 105°C
  • Preheated to 98°C
    • Phase °C Time (sec)
    Initial Denaturation 98 30
    Denaturation 98 10
    Annealing 67 30
    Extension 72 30
    Final Extension 72 120
    Hold 4 Held
  • 2-3 mL LB cultures of YEP352GAP
    • e. coli prepped from each
    • Restreaking from 7/18
  • 3mL of LB Broth with 3mL Ampicillin
  • Control set up with RFP Plasmid e. coli
  • Incubated at 37°C at 3:12am - 220rpm
Gel run on 7/20 Q5 PCR Product

Lane:

  1. Ladder
  2. Rx #1 5ul + 1ul dye
  3. Rx #2 5ul + 1ul dye
Q5 PCR of PCR Template (Phire - 25ul Rx)
    Contents Amount (ul)
    H2O 9.5
    Master Mix 12.5
    10uM F Primer 1.25
    10uM R Primer 1.25
    Template (95.1ug/ul) .5
    • Thermocycler set up to the same specifications as earlier today, except the annealing temperature is now 68°C
    • Prepped a gel for running PCR Reactions
    "I broke up with someone over AIM" -Sarah


7/21

Miniprep of Dean Vector using 2-3ml

  • LB culture made 7/20
  1. Centrifuged at 4500rpm for 2 minutes and 30 seconds
  2. Centrifuged at 4500rpm for 5 minutes, lysed LB cultures
  • Followed NEB protocol from then on

Two gel runs

  • Ran two consecutive gels with Reactions 1 and 2
  • Gels ended up smearing
    • Make a serial dilution of template
    • Too many cycles (Maybe lower it to 25-30)
    • Primer concentration not optimal
3 Reactions set up with Q5 2X Master Mix Polymerase (25ul Rx)
    Content Rx #1 Rx #2 Rx #3
    H2O 9.9 9.7 9.5
    10X uM F Primer 1.25 1.25 1.25
    10X uM R Primer 1.25 1.25 1.25
    Q5 Master Mix 12.5 12.5 12.5
    95.1 ug/ul Template 0.1 0.3 0.5
  • Machine set up normally except the annealing temperature set up for 67°C
  • Ran at 28 cycles
"Failure is a metaphor" -Samara


7/22

Set up PCR Rx for construct (25ul Rx x2 - 28 cycles)

    Content Rx1 (ul) Rx2 (ul)
    H2O 9.5 9.9
    10uM F Primer 1.25 1.25
    10uM R Primer 1.25 1.25
    95.1 ug/ul Template 0.5 0.1
    Q5 Master Mix 12.5 12.5
  • PCR run at normal settings except the annealing temperature is now 52°C