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Team:DTU-Denmark/Notebook/22 July 2013
From 2013.igem.org
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===USER reaction of AMO and HAO with pZA21 (with native promoter)=== | ===USER reaction of AMO and HAO with pZA21 (with native promoter)=== | ||
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| - | USER reaction was performed according to standard protocol (***). | + | USER reaction was performed according to standard protocol but using more fragment because of the low DNA concentration in the PCR purification of the fragments (***). |
USER mix: | USER mix: | ||
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Incubation of cells on ice for 30 minutes, heat shock at 42C for 90 sec. and 5 min on ice. Addition of SOC medium and 2 hours of incubation at 37C. Plating on plates with LB medium with Kanamycin, overnight incubation at 37C. | Incubation of cells on ice for 30 minutes, heat shock at 42C for 90 sec. and 5 min on ice. Addition of SOC medium and 2 hours of incubation at 37C. Plating on plates with LB medium with Kanamycin, overnight incubation at 37C. | ||
| + | ===PCR of AMO and HAO with USER endings=== | ||
| + | |||
| + | Performed PCR to extract AMO and HAO from nitrosomonas europeae as the amount of purified fragment is getting lower. Settings according to previously successful PCRs were: | ||
| + | |||
| + | AMO: 54C, 3 mins | ||
| + | HAO: 56C, 3 mins | ||
| + | |||
| + | |||
| + | ==Results== | ||
| + | |||
| + | Run 1% agarose gel to analyze products on the PCR for AMO and HAO performed today. AMO is not observed and instead of HAO we receive and unknown fragment of length ~300 bp. | ||
{{:Team:DTU-Denmark/Templates/EndPage}} | {{:Team:DTU-Denmark/Templates/EndPage}} | ||
Revision as of 17:09, 22 July 2013
22 July 2013
Contents |
208
Main purpose
- ON culture of Colony PCR products of HAO and AMO from 19-07-2013
- USER reaction of AMO and HAO with pZA21 (with native promoter).
- RFP amplification in pZA21 using primers without promoter
Who was in the lab
Henrike, Kristian, Julia
Procedure
USER reaction of AMO and HAO with pZA21 (with native promoter)
USER reaction was performed according to standard protocol but using more fragment because of the low DNA concentration in the PCR purification of the fragments (***).
USER mix:
- USER enzyme 1 uL
- NEB 4 buffer 0,5 uL
- 10x BSA 0,5 uL
- backbone pZA21 1uL
- Sample 1 -> 3 uL of USER mix + 12 uL of AMO
- Sample 2 -> 3 uL of USER mix + 12 uL of HAO
- Sample 3 -> Negative control, USER mix + 12 uL water
USER reaction was performed in PCR machine with programm: 37C for 40 min and then 25C for 30 min.
To 100uL of chemically competent E. coli cells 10 uL of USER mix after USER reaction was added.
Incubation of cells on ice for 30 minutes, heat shock at 42C for 90 sec. and 5 min on ice. Addition of SOC medium and 2 hours of incubation at 37C. Plating on plates with LB medium with Kanamycin, overnight incubation at 37C.
PCR of AMO and HAO with USER endings
Performed PCR to extract AMO and HAO from nitrosomonas europeae as the amount of purified fragment is getting lower. Settings according to previously successful PCRs were:
AMO: 54C, 3 mins HAO: 56C, 3 mins
Results
Run 1% agarose gel to analyze products on the PCR for AMO and HAO performed today. AMO is not observed and instead of HAO we receive and unknown fragment of length ~300 bp.
