July 1st

What to do:

• Resuspend plasmids from the iGEM kit
• Transform to E.coli cell
• Store the remaining suspended plasmid

Reagents and equipments

• Reagents
  1. Distilled water
  2. Ice
  3. 50 µL competent cells for parts of choice
  4. 50 µL competent cells for positive control
  5. Positive control
  6. SOC Media
  7. Agar plates with appropriate antibiotic

     § NOTE: this needs to be prepared beforehand.

• Equipments
  1. 10 and 200 µL pipettes
  2. Timer
  3. Water bath (420C)

     Procedures

• Resuspending the part
  1. Mark the location of the parts using a pen or marker
  2. Punch a hole through the foil using a pipette tip
  3. Add 10µL of dH2O
  4. Mix thoroughly by aspirating up and down a few times
• Inoculating the DNA
  1. Add 1µL of the suspended DNA to the competent cells

     § The competent cells must be kept on ice before and after the addition of the suspended DNA § Make sure to mark the lid of the eppendorf tube, which tube is filled with C.C +DNA and which tube is filled with positive control.

  2. Add positive control to the competent cells

     § ASK: What strain of E.coli are we going to use as the competent cell? E.coli codon plus? Top10? BL21? pLysS?

• Incubate on ice for 30 minutes
• Heatshock in water bath for 1 minute
• Incubate on ice for 5 min
• Add 200 µL of SOC media to each transformation

  § ASK: Is it available in IHVCB? If SOC media is not available, can we replace it with regular LB broth?

• Incubate for 2 hours at 370C
• Plate out the tranformation
  1. Plate 20 µL to one agar plate, and the remainder to the separate plate.
  2. Use beads to spread evenly
  3. Incubate overnight (16hrs) at 370C

     § ASK: What antibiotics do we need? Is it available in IHVCB?

• Store the remaining resuspended parts

  § ASK: In what temperature do we keep the resuspended part?

July 7th

• Making 20 plates of LB Agar with Chlorampenicol added (Chloramphenicol concentration = 25 μg/mL

July 9th

• Resuspending the parts we need
• Transforming the plasmid into E.coli top 10 cells
• Spread the transformed cells into LB Agar Plates containing appropriate antibiotics

July 10th

- No growth in positive control plate for ampicillin - Growth in negative control on chloramphenicol - No growth on RBS plate ○ Possiblity: contamination - Miniprep for isolation ○ 2 tubes (orange cap) @4mL broth + 4μL amp ○ 5 tubes (blue c ap) @4mL broth + 2 μL chl ○ 5 tubes (blue cap) @4mL broth + 2 μL IHVCB chl

July 11st

Today's to do list:

• Running electrophoresis

  ○ CHECK THE PROTOCOL

• Prepare another agar plates (chloramphenicol ..., ampicilin ...)
• Re-transform!

  ○ ASK: what's wrong with the other plates? Wrong dosage of antibiotics? Contamination?

  § Check the concentration of the chloramphenicol needed for each plate

  □ Checked, 1: 2000 § The previous agar plates only has 1:4000 concentration of antibiotics § The previous antibiotic was diluted in H2O, while there is a source which mentions that they use 25 mg/mL chloramphenicol diluted in 100% ethanol § Possible reason as to why extreme growth are observed in chloramphenicol plates ○ Prepare agar plates for 5 chloramphenicol resistant plasmids and 2 ampicillin resistant plasmids, plus positive and negative control for each antibiotic.

  § Plates needed: 10 + 2 + 4 + 2 = 18 plates -> make 20, + 2 amp -> 12:8 § 240mL agar for chloramphenicol, 160 mL agar for ampicilin

What we did:

• Make another 6 plates of agar +amp and 12 plates agar +chl (+1 plate +amp for kak Gema)
• Isolating the plasmids from transformed E.coli
• Running the plasmid (failed)

July 12nd

• Electrophoresis iGEM plasmid

July 15th

• Re-transform the plasmid

July 16th

• Checking the plates -> contamination
• Making new agar plates

July 17th

• Primer designing
• Preparing to make competent cells

July 22nd

- Re-run Well 1: marker Well 2: alfa 1/2 (colony/transform) Well 3: alfa 2/2 Well 4: full 1/2 Well 5: full 2/2 Well 6: alfa 1/3 Well 7: alfa 2/3 Well 8: full 1/3 Well 9: full 2/3

July 23rd

• Restricting plasmids
• Running

July 24th

- Re-digest the plasmids especially containing lacZ full - Re-run - Make maxiprep

List of materials

• Digesting plasmids
  • NEBuffer 10x
  • BSA 10x
  • H2O sigma (DW)
  • EcoRI (-800C freezer)
  • DNA
• Running
  • Agarose
  • LD 6x
  • Marker

July 25th

• Large scale isolation for LacZfull
• Inoculating RFP

July 26th

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Running LacZ full and lacZ alpha

Inoculating lacZ alpha

Find restriction site with buffer compatible with EcoRI XbaI SpeI and PstI

July 27th

• Large scale isolation lacZ alpha
• Making agarose
• Electrophoresis
  • From the UV reading of the agar, we found that the plasmid is transformed into the cell. We decided to linearize the plasmid

July 30th

Labwork to do list:

• Digesting lacZ alpha
• Running

July 31st

• Linearizing lacZ alpha using EcoRI
• Electrophoresis
  • We got the correct length for lacZ alpha

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