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Lab notes (7/26 - 7/30)

Contents 1 Lab notes (7/26 - 7/30) 1.1 Group: Flagella 1.1.1 Incertion of Promoter + RBS in pSB3T5 1.1.1.1 Restriction and Gel extraction 1.1.1.2 Purification of J13002 and pSB3T5

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.

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:

Tubes	1P (Plasmid)	2P	3B (Brick)	4B
Tube weight	1g	1g	1g	1g
Gel weight	170mg	110mg	245mg	160mg

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Tube	Concentration (ng/ul)
1P	9.6
2P	2.9
3B	6.7
4B	3.2