Team:Kent/Notebook

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Logbook

Project Logbook

Week 5

• Order of IDT gBlocks and primers
• Transformation of pSB1C3 + GFP into DH5α cells
• Plated and O/N growth at 37°C
• Next day, inoculate LB overnight cultures
• Miniprep DNA

Week 6

• Order of gRNAs
• Digest (GFP +pSB1C3) DNA with ECO R1 and Pst1 to confirm presence of plasmid (Fig 1)
  • DNA marker ( 1kb DNA life plus)
  • Undigested plasmid (pSB1C3 containing GFP)
  • Digested with Eco RI
  • Digested with PstI
  • Digested with both, ECO RI and PstI

Week 7

Amplification of dCas13a failed – order of new primers

Week 8

Competent DH5α cell preparation

Week 9

Amplification of dcas13a

• DNA marker (1kb DNA life plus) -- Lane 1
• dCas13a (3477 bp) -- Lane 3
• dCas13a (3477 bp) -- Lane 4

Week 10

• Resuspension of gRNA
• Gibson assembly of pSB1CR + GFP with dCas13a
• Transformation and plated, incubated at 37°C overnight
• Set up LB overnights, patch plate and mini prep
• Digestion of whole plasmid with EcoR1 and Pst1 to check for band sizes (Fig 3)

Amplification of plasmid (whole gBlock + Backbone) with screening primers

Week 11

Successful amplification gBlock1

• Screening gel, complete construct PCR amplified with screening primers whole construct ~6600 bp
  • DNA 1 Kb ladder -- Lane 1
  • pSB1C3 containing GFP and dCas13a -- Lane 3-5x

Week 12

• PCR amplify gBlock2 (dCas13a) and 1% agarose gel to confirm – failed
• Design and order of new dCas13a primers

Week 13

Run 1% agarose gel of gBlock 2 PCR products and pcDNA 3.1 vector amplification

• Once bacterial growth has been achieved, 500μL of the overnight liquid culture needs to be added to 500μL of 50% glycerol in a 2mL tube where it should be gently mixed
• The glycerol stock should then be frozen at -80 o C
  • Successive freeze and thaw cycles will reduce the stocks shelf life