Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/03/18

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Overview

The goal of this project is to change the reporter gene in a stably transfected cancer cell line. The current reporter gene is luciferase, which has poor resolution on a cellular level and requires a time-intensive assay to measure. We intend to replace the luciferase gene and promoter with cyan fluorescent protein (CFP) for rapid measurement of reporter gene activity. We'll also be replacing the luciferase TK promoter with a HPK promoter, which should enhance transcription.

Here's the overall direction this project is going to take:

  1. Order a gBlock from IDT containing the two homology arms for switching out TK-luciferase, restriction sites for inserting the gBlock into a cloning plasmid, and a Type IIS restriction site between the homology arms for inserting the HPK promoter and the CFP gene.
  2. Order primers from IDT for adding an overhang containing a Type IIS restriction site to A) the CFP gene fragment; B) the HPK promoter fragment.
  3. Traditional restriction digest and ligation of cloning plasmid and gBlock to insert the gBlock into the plasmid.
  4. Single pot digestion and ligation using Type IIS restriction endonuclease to combine the HPK promoter and CFP into the middle of the gBlock inside the cloning plasmid.
  5. Mini-prep the plasmid to make many copies, cut out and isolate our fused product.
  6. CRISPR to insert the product into the mammalian cell line, replacing TK-luciferase.
  7. FACS to enrich for successfully transfected cells.
  8. Isolate single colonies of stably transfected cells, grow up, and make stocks.

DBN002 & DBN003 PCR

Primers and gBlock have already been ordered, and primers arrived yesterday. Today I made 100μM stocks of the primers and used the primers to amplify the HPK sequence (with Kozak) and the CFP sequence.

PCR kit used was GoTaq clear 2x Master Mix, containing polymerase, buffer, and dNTPs.

Reagent Volume (µL)
2x master mix 25
MB grade H2O 24
forward primer 0.25
reverse primer 0.25
template 0.5

Four reactions were performed. Rxn 1 used KAH45 plasmid as the template (from BioBricks box) and DBN002 f1 and DBN002 r1 as the primers. Rxn 2 used KAH104 plasmid as the template (from BioBricks box) and DBN003 f1 and DBN003 r1 as the primers. Each reaction also had a control reaction containing primers but no template.

PCR program used is named DBN031815. 30 cycles were performed.

Step Temperature °C Duration
Initial denature 95 120 sec
denature 95 30 sec
anneal 59 / 68 30 sec
extension 72 90 sec
final extension 72 300 sec
soak 4 indef.

The DBN002 primers anneal at 59°C and the DBN003 primers anneal at 68°C.