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Team:TorontoMaRSDiscovery/Notebook
From 2009.igem.org
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| - | + | =2009-01-27= | |
| + | Received from Rosa (SPiT) | ||
| + | <Br /> | ||
| + | |||
| + | *TM0785 | ||
| + | **Plasmid containing encapsulin | ||
| + | **Recommend transfect into bacteria and re-sequence | ||
| + | **See email note regarding sequence error | ||
| + | |||
| + | *0.5 microliters TMG DNA 100 microgram/microliter | ||
| + | **Use 0.4 microliter for 50 microliter PCR reaction | ||
| + | **This is thumotoga maritime genomic DNA for purpose of re-cloning | ||
| + | |||
| + | Microcentrifuge tubes 1 and 2 placed in -20 freezer | ||
| + | |||
| + | 2009-05-15 | ||
| + | 1. pH 4 buffer (red) 500 ml | ||
| + | pH 7 buffer (yellow) 500 ml | ||
| + | pH 10 buffer (blue) 500 ml | ||
| + | Above are used for pH/mV Meter calibration | ||
| + | 2. pH/mV meter calibrated according to manual – recorded in index | ||
| + | 3. Ethanol solution (70%) made from 85% ethanol | ||
| + | |||
| + | 2009-05-19 | ||
| + | 1. 2L of TE buffer made (10X TE) | ||
| + | a. Recipe for 2L from stock solution (10X TE) | ||
| + | i. 10 ml 1M Tris-HCl pH 7.5-80.0 x 2 = 20 ml | ||
| + | ii. 2 ml 0.5 M EDTA pH 8.0 x 2 = 4 ml | ||
| + | iii. 988 ml ddH20 x 2 = 1976 ml | ||
| + | |||
| + | - To make Tris-HCl, Tris base is used and adjusted to desired pH using HCl | ||
| + | - 1 M Tris base is required of which 20 ml is required – thereby 2.4228 g Tris base (due to scale limitations, 2.42g of Tris Base was used | ||
| + | |||
| + | 2. 500 ml of 1 M Tris Base made | ||
| + | - mass of Tris used = 1M/1000 ml x 500 ml x 121.14 g/mol = 60.57 g | ||
| + | - volume of water used = 500 ml | ||
| + | |||
| + | 3. 250 ml of 0.5 M EDTA solution was made | ||
| + | - mass of EDTA used = 36.53 g | ||
| + | - observations: EDTA did not dissolve in ddH2O on heat and being stirred | ||
| + | |||
| + | 10 MINUTE BRIEFING (INSERT) | ||
| + | |||
| + | 2009-05-21 | ||
| + | |||
| + | - retrieved autoclaved ddH20, glycerol solution | ||
| + | |||
| + | Gel Electrophoresis (test run) | ||
| + | |||
| + | - 1% gel: 0.5 g agar mixed with 50 ml of 1X TBE | ||
| + | - 10X TBE diluted with 5 ml TBE + 45 ml ddH20 – did not use all 50 ml for 1 gel (gel will be too thick – make 2 instead) | ||
| + | - Loading Dye: add glycerol solution to 25 mg bromophenol blue then add dH20 to 10 ml (to make 6X) | ||
| + | - Running gel: match wells to black side, run at 120 mA | ||
| + | |||
| + | Visualize Gel in UV | ||
| + | 1. Turn power on | ||
| + | 2. Gel in machine face up | ||
| + | 3. Close door securely | ||
| + | 4. Turn white light on | ||
| + | 5. Adjust zoom, contrast, focus from black dial on top of machine | ||
| + | 6. Turn white light off (turns on UV) | ||
| + | 7. Press ‘live’ toggle – acq. Should be 0.4 sec. | ||
| + | 8. Print if desired or save on floppy disk | ||
| + | 9. Turn power off | ||
| + | 10. Dispose of gel in proper container | ||
| + | 11. Close door | ||
| + | |||
| + | 2009-05-25 | ||
| + | |||
| + | - made overnight bacteria culture (200 ml LB: 20 microliters DB3.1) | ||
| + | |||
| + | 2009-05-26 | ||
Revision as of 14:40, 4 September 2009
2009-01-27
Received from Rosa (SPiT)
- TM0785
- Plasmid containing encapsulin
- Recommend transfect into bacteria and re-sequence
- See email note regarding sequence error
- 0.5 microliters TMG DNA 100 microgram/microliter
- Use 0.4 microliter for 50 microliter PCR reaction
- This is thumotoga maritime genomic DNA for purpose of re-cloning
Microcentrifuge tubes 1 and 2 placed in -20 freezer
2009-05-15 1. pH 4 buffer (red) 500 ml pH 7 buffer (yellow) 500 ml pH 10 buffer (blue) 500 ml Above are used for pH/mV Meter calibration 2. pH/mV meter calibrated according to manual – recorded in index 3. Ethanol solution (70%) made from 85% ethanol
2009-05-19 1. 2L of TE buffer made (10X TE) a. Recipe for 2L from stock solution (10X TE) i. 10 ml 1M Tris-HCl pH 7.5-80.0 x 2 = 20 ml ii. 2 ml 0.5 M EDTA pH 8.0 x 2 = 4 ml iii. 988 ml ddH20 x 2 = 1976 ml
- To make Tris-HCl, Tris base is used and adjusted to desired pH using HCl - 1 M Tris base is required of which 20 ml is required – thereby 2.4228 g Tris base (due to scale limitations, 2.42g of Tris Base was used
2. 500 ml of 1 M Tris Base made - mass of Tris used = 1M/1000 ml x 500 ml x 121.14 g/mol = 60.57 g - volume of water used = 500 ml
3. 250 ml of 0.5 M EDTA solution was made - mass of EDTA used = 36.53 g - observations: EDTA did not dissolve in ddH2O on heat and being stirred
10 MINUTE BRIEFING (INSERT)
2009-05-21
- retrieved autoclaved ddH20, glycerol solution
Gel Electrophoresis (test run)
- 1% gel: 0.5 g agar mixed with 50 ml of 1X TBE - 10X TBE diluted with 5 ml TBE + 45 ml ddH20 – did not use all 50 ml for 1 gel (gel will be too thick – make 2 instead) - Loading Dye: add glycerol solution to 25 mg bromophenol blue then add dH20 to 10 ml (to make 6X) - Running gel: match wells to black side, run at 120 mA
Visualize Gel in UV 1. Turn power on 2. Gel in machine face up 3. Close door securely 4. Turn white light on 5. Adjust zoom, contrast, focus from black dial on top of machine 6. Turn white light off (turns on UV) 7. Press ‘live’ toggle – acq. Should be 0.4 sec. 8. Print if desired or save on floppy disk 9. Turn power off 10. Dispose of gel in proper container 11. Close door
2009-05-25
- made overnight bacteria culture (200 ml LB: 20 microliters DB3.1)
2009-05-26
