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Team:SDU-Denmark/labnotes3
From 2010.igem.org
Lab notes (7/26 - 7/30)
Contents |
Group: Flagella
Incertion of Promoter + RBS in pSB3T5
Done by: Christian and Louise
Parts used: J13002 (promoter+RBS) and pSB3T5
Restriction and Gel extraction
Protocol: [RD1.1]
Notes: We made 2 Restriction mixtures which both were 4 times the mixture in the protocol. We also added 10 ul less water and 10 ul more PCR product than discribed in the protocol
Restriction mixture
- 38 ul Water
- 4 ul EcoRI enzyme
- 4 ul PstI enzyme
- 8 ul Fast Digest green buffer
- 30 ul PCR product (Freeze tube white 25 PROMOTER + RBS)
OR
- 30 ul PCR product (Freeze tube white 29 PLASMID)
Loading:
2 x 21 ul J13002 was loaded on a 2% gel with a 100-1000bp ladder. The Restricted J13002 is 95bp.
2 x 21 ul pSB3T5 was loaded on a 1.5% gel with a 100-10,000 ladder. The restristricted plasmid is 3215bp.
Result:
The picture shows a band around 100bp.
The picture showes two bands, one around 3000bp which is the plasmid and one around 1000bp, which is RFP.
Purification of J13002 and pSB3T5
Protocol:
Notes: four tubes were marked and weighed (all had a mass of 1g). Bands were cut from the restriction gels, added to the tubes and weighed.
| Tubes |
1P (Plasmid) |
2P |
3B (Brick) |
4B |
| Tube weight |
1g |
1g |
1g |
1g |
| Gel weight |
170mg |
110mg |
245mg |
160mg |
Sample capture: 300 ul Capture buffer type 3 was added to the tubes and the tubes were placed at 60 degrees for 20 minutes.
Sample binding:The Capture buffer sample mix were added to MicroSpin Columns, stood for 1 minute and centrifuged for 30 sec. at 16,000g.
The Wash and Dry was carried out according to the protocol.
Elution: 10 ul Elution buffer type 6 was added to the columns, stood for 1 minute and centrifuged for 1 minute at 16,000g. The columns were thrown out and NanoDroped.
NanoDrop:
| Tube |
Concentration (ng/ul) |
| 1P |
9.6 |
| 2P |
2.9 |
| 3B |
6.7 |
| 4B |
3.2 |
