Team:TP-CC San Diego/LabBook

Lab Notebook

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SUMMARY of July 17th:

  • DNA extraction from GBM39 cells
  • Agarose gel electrophoresis to confirm DNA


SUMMARY of July 18th:

  • qPCR primer design and order


SUMMARY of July 21:

  • Primers arrived and diluted according to instructions


SUMMARY of August 1st:

  • qPCR for EGFR gene count and guide RNA design; placed order


SUMMARY of August 2nd:

  • Received oligos and diluted according to instruction.


SUMMARY of August 7th:

  • Digestion of lentiviral CRISPR plasmid
  • Gel purification digested plasmid using QIAquick Gel Extraction Kit and eluted in buffer EB
  • There should be two bands on the gel, one at 2kb which should not be purified. Purify only the larger band


SUMMARY of August 9th:

  • Anneal oligo pairs
  • Ligation and transformation


SUMMARY of August 10th:

  • No colonies grew on carb antibiotic plates


SUMMARY of August 11th:

  • Used same,remaining ligation reaction and transformed again onto carb antibiotic plates

DAY 10

SUMMARY of August 14th:

  • Inoculate comp cells

DAY 11

SUMMARY of August 15th:

  • Miniprep gRNA plasmids and sent in for sequencing
  • Start interlab study
  • Ludox Calibration
  • Fluorescein Dilution
  • Transform plasmids from plate 7 in kit provided by IGEM for interlab study

DAY 12

SUMMARY of August 16th:

  • Received sequencing results and all plasmids have the gRNA
  • Inoculate plasmids for interlab study

DAY 13

SUMMARY of August 17th:

  • Interlab cell measurement

DAY 14

SUMMARY of August 21st:

  • seed ~1.2 x 107 293T cells in a 10 cm dish

DAY 15

SUMMARY of August 17th:

  • Prepared the transfection complex.

DAY 16

SUMMARY of August 23rd:

  • Removed the transfection medium from cells
  • Miniprep plasmids and measure concentration

DAY 17

SUMMARY of August 24th:

  • Harvest cells

DAY 18

SUMMARY of August 25th:

  • Plate target cells, incubate overnight

DAY 19

SUMMARY of August 26th:

  • Remove the cell culture medium and replace with fresh medium, incubate overnight

DAY 20

SUMMARY of August 27th:

  • Split cells 1:3 and incubate for 48 hours

DAY 21

SUMMARY of August 29th:

  • Infected cells are selected for stable expression
  • Continue monitoring cells

DAY 22

SUMMARY of October 21st:

  • Digest guideRNA using restriction enzymes out of TLCV2 vector
  • Run 1% agarose gel and cut out insert
  • Gel purify insert and vector(pSB1C3) given by iGEM
  • Ligation of guide RNA insert and pSB1C3 vector
  • Transformation of plasmid for part submission

DAY 23

SUMMARY of October 22nd:

  • Inoculated plasmid for part submission

DAY 24

SUMMARY of October 23st:

  • Miniprep plasmids and measure concentration