Team:Kent/Notebook

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Week 1

Overnight cultures of DH5α E.coli cells
Prepared competent E.coli cells and tested them with the iGEM competent cell test kit + results
- Images of patched plates (?)
- Bar chart of competent cells
- Competency of cells - numbers are: 10pg/μl: 5 and 8; 50pg/μl: 35 and 5; 100pg/μl 39 and 104
Prepared LB medium, SOC medium, SOB medium and autoclaved materials

Week 2

Made glycerol stocks of competent DH5α E. Coli overnights (1:1) with 50% glycerol stock and store at -80 C (keep for years)
DNA mini prep PSB1C3 (Qiagen)
Restriction digest using ECO R1 from Promega
Run Gel of digested samples to confirm presence of desired plasmid (compare fragment sizes)
Concentration of our DNA is 90 ng/mL (Nanodrop?)

Chloramphenicol plates to test whether plasmid (containing antibiotic resistance gene) was incorporated into bacteria
Before----------------------After

Did head shots in the green room

Week 3

Transformation of GFP into Backbone (BBa_K125500 into PSB1A3) + grow overnight in LB medium
DNA mini prep (Qiagen)
Agar gel (1%) with 2 digestion enzymes (Eco R1 and Pst I)
Talked to Dr. Garrett about mammalian cell use
Ordered 3 different Cas13a Plasmids containing different mutations in the two HEPN domains

Week 4

Our Plasmids arrived (Yaas!)
Nanodrop of Week 3 GFP transformation into PSB1A3 vectors (2 attempts, low but gels were ok)
Resuspension of stabs and overnight growth of (all three) cultures in LB medium
Digestion with Eco R1 of all 6 samples (each twice = 3x2)
0.7% Agarose Gel to confirm presence of plasmid
Talked to Dr. Ellis, Dr. Kad & Taylor about Gibson assembly and Primer design
Designed primers in Genome Compiler

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

Week 14

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

Week 15

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

Week 16

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