"
Team:DTU-Denmark/Notebook/12 July 2013
From 2013.igem.org
(→USER reaction of RFP and pZA21 (with native promoter) and transformation of E. coli cells) |
(→USER reaction of RFP and pZA21 (with native promoter) and transformation of E. coli cells) |
||
| Line 79: | Line 79: | ||
To 100uL of chemically competent ''E. coli'' cells 10 uL of USER mix after USER reaction was added. | 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. | + | 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. |
Revision as of 16:26, 12 July 2013
208
Main purpose
- miniprep of biobrick transformants from 10-07-2013
- PCR with USER primers to amplify cytochromes, AMO, HAO and Nir genes
- gel analysis of PCR products from Nir operon, AraBAD promoter and RFP
- USER reaction of RFP and pZA21 (with native promoter) and transformation of E. coli cells
Who was in the lab
Henrike, Julia, Kristian, Jakob, Gosia
Procedure
Plasmid isolation of biobricks from transformants made on 10-07-2013
Isolation was performed according to protocol given in the kit PowerPrep HP Plasmid Miniprep Kit
Amplification of cytochromes, AMO, HAO and Nir genes
Each PCR reaction was performed in triplicate.
Samples are named:
- 1, 2, 3 -> cytochromes
- 4, 5, 6 -> AMO
- 7, 8, 9 -> HAO
- 10, 11, 12 -> Nir
PCR reaction mix was done according to standard procedure.Primers and tamplates used for samples are as follows:
- 1, 2, 3 -> cytochromes, primers 16a, 16b, template CYC isolated by colony PCR from Nitrosomonas europea
- 4, 5, 6 -> AMO, primers 17a, 17b, template AMO isolated by colony PCR from N. europea
- 7, 8, 9 -> HAO, primers 18a, 18b, template HAO isolated by colony PCR from N. europea
- 10, 11, 12 -> Nir, primers 15a, 15b, template - 2uL of liquid culture of "Pseudosomonas"
PCR programs based on standard program with differences in annealing temperature and elongation time as follows:
- 1, 2, 3 -> cytochromes - 57°C, 1:30 min
- 4, 5, 6 -> AMO - 54°C, 3 min
- 7, 8, 9 -> HAO - 59°C, 9 min
- 10, 11, 12 -> Nir - 59°C, 9 min
Gel analysis of PCR products from Nir operon, AraBAD promoter and RFP
1 % Agorase Gel (Nir operon)
Wells
- 1: ladder 1kb
- 2: RFP
- 3: RFP
- 4: AraBAD promoter
- 5: AraBAD promoter
- 6: AraBAD promoter
- 7: RFP
- 8: Nir
- 9: Nir
- 10: Nir
- 11:Nir
- 12: Nir
- 13: Nir
- 14: Nir
- 15: Ladder 1 kb
USER reaction of RFP and pZA21 (with native promoter) and transformation of E. coli cells
USER reaction was performed according to standard protocol.
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 + 7 uL of RFP
- Sample 2 - Negative control, USER mix + 7 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.
