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30 June 2009
From 2009.igem.org
Results of 29 June 2009
| Number | Part Name | Content(length/bp) | Backbone(length/bp) | Location | Plate Result |
| 18 | R00405 | TetR_Promoter(???) | pSB1A2(2079)) | P1_06I | √(only 2 colonies) |
| 19 | Q04400 | RBS+TetR_Coding+T+TetR_Promoter(???) | pSB1K3(???) | P1_16P | X |
Aim:
- Picking 2 colonies from plate 16; plate 18 for incubation.
- Transformation of NO.18 & 19 again.
- Picking 102 colonies from plate 9(contaminated by fungi) for incubation.
Materials:
•TE Buffer
•Plasmids from Kit Plates 1: R0040; Q04400
•Competent E.coli
•NZY Medium
•Ampicllin, Kanamycin Agar Plates
•Eppendorf (labelled)
Methods:
- Tranformation:
- Add 7µL of 50ºC TE buffer into R0040well & Q04400well to make the total volumn upto 10µL.
- Add 5µL of DNA in TE to 50µL of competent cells (TOP10)
- Allow the DNA and competent cells to sit on ice for 30 minutes
- Heat shock at 42ºC for 60 sec in water bath.
- Recover on ice for 5 min.
- Add 300 µL NZY medium.
- Incubate at 37ºC for 2 hr while the tubes are rotating.
- Centrifuge and leave about 250 µL liquid; hence, resuspend the E.coli suspension.
- Plate 100µL on an LB plate with the appropriate antibiotic.
- Incubation:
- Pick new colonies from plate 9 (FGHIJKLMNO), plate 16(ABCD) & 18(AB). <Since Tube 16AB showed no growth after about 7 hrs incubation, another two colonies(CD) were picked at 17:00>
- Incubate with shaking overnight.
