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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 |
The Concentrations were quite low, but we pooled them (1P+2P and 3B+4B) and used them for ligation.
Follow-up colony PCR
Date: 26/7
Methods: Colony PCR
Protocol: CP1.3
Experiment done by: Maria, LC
Notes: We made a sample for every plate from the last colony PCR. Out of the 29 samples we could only run 25 at once in the PCR machine. Plate 23 was missing, so there is no sample for it.
Results: 
Lengths of PCR products:
1200: 4, 5, 6, 9, 10, 14, 21, 26
1500: 13
1750: 2
1900: 8, 25
2000: 3, 7, 12, 18, 19, 24
2200: 20
2500: 11, 17
Colonies 1, 15, 16 and 22 gave no result.
Analysis: Since we had so many different lengths, we will cut one from each length, specifically colony: 2, 8, 11, 13, 20, 24, 26.
Miniprep of follow-up colony PCR
Date: 27/7
Methods: ON, Miniprep
Protocol: MP1.1
Experiment done by: LC
Notes: Samples L4, L5, L6, L9, L10, L14, L21 and L26 (L for ligation from the follow-up colony PCR) miniprepped and NinaB samples from the 4 frozen cultures of it.
Results: 
All ligations were around the same length, just under 2000 BP. Even though the correct length should have been about 1000 BP longer, this is not surprising since uncut plasmids often show 1000 BP less in length. NinaB was also too short and there was a second band showing higher up in the gel (weird!). The concentrations averaged around 80ng/ul for the samples.
Analysis: We decided to do a new miniprep where we start with a higher concentrated overnight culture, which we will achieve through boosting the ON before doing the miniprep.
Digestion and gel extraction of pSB3C5 and J13002
Done by: Pernille
Date: the 26th of july
Protocol: [RD1.1] and gelpurificaton (DE1.3)
