Team:UI-Indonesia/July
From 2013.igem.org
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<h1><span id= "The_Great_Team"; style="color:white";>July</span></h1> | <h1><span id= "The_Great_Team"; style="color:white";>July</span></h1> | ||
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<h1>July 1st </h1> | <h1>July 1st </h1> |
Latest revision as of 02:41, 19 October 2013
July
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
- Distilled water
- Ice
- 50 µL competent cells for parts of choice
- 50 µL competent cells for positive control
- Positive control
- SOC Media
- Agar plates with appropriate antibiotic
§ NOTE: this needs to be prepared beforehand.
- Equipments
- 10 and 200 µL pipettes
- Timer
- Water bath (420C)
- Procedures
- Resuspending the part
- Mark the location of the parts using a pen or marker
- Punch a hole through the foil using a pipette tip
- Add 10µL of dH2O
- Mix thoroughly by aspirating up and down a few times
- Inoculating the DNA
- 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.
- 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?
- Add 1µL of the suspended DNA to the competent cells
- 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
- Plate 20 µL to one agar plate, and the remainder to the separate plate.
- Use beads to spread evenly
- 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
ul>
- 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
- Digesting lacZ alpha
- Running
Labwork to do list:
July 31st
- Linearizing lacZ alpha using EcoRI
- Electrophoresis
- We got the correct length for lacZ alpha