"
Team:Groningen/Labwork/22 July 2013
From 2013.igem.org
| Line 14: | Line 14: | ||
Run a gel with the ligation product of silk and the PCR afterwards (made on 19-07). Figured out why the ligation did not work. | Run a gel with the ligation product of silk and the PCR afterwards (made on 19-07). Figured out why the ligation did not work. | ||
| - | Made a new restriction digest for silk without strep tag. | + | Made a new restriction digest for silk without strep tag. Load it on gel and saw a very small band. The concentration was 52.2 ng/ul, which is surprisingly high. So a ligation is made with all four signal sequences. |
'''Inne''' | '''Inne''' | ||
Revision as of 13:04, 22 July 2013
Mirjam
Made a PCR for silk 2 and 3 (strepF-R and F-strepR)in triplo
- 28 ul MQ
- 1.5 ul DMSO
- 10 ul GC buffer
- 1 ul of each dNTP
- 2.5 ul primer F
- 2.5 ul primer R
- 0.5 ul phusion
- 1 ul genomic DNA (BBa_...016)
Run a gel with the products to determine if the silk is present. Silk is present in high amounts, but only for the parts under 500 bp. That is why a new PCR is made for both silks.
Run a gel with the ligation product of silk and the PCR afterwards (made on 19-07). Figured out why the ligation did not work.
Made a new restriction digest for silk without strep tag. Load it on gel and saw a very small band. The concentration was 52.2 ng/ul, which is surprisingly high. So a ligation is made with all four signal sequences.
Inne
Ran Gel to check everything that was in the freezer before discarding it.
Gel was 1.5% and ran at 90V for 45 min
Checked tubes:
| Slot | Description Lid | Description Side |
| 1 | 1kb generuler | Fermentas 1kb DNA Ladder (and more) |
| 2 | 1 | Silk 4.1ng/uL Strep R-F |
| 3 | 1 | Silk 4.1ng/uL F-R |
| 4 | 2 | Silk 4.6ng/uL Strep R-F |
| 5 | 2 | Silk 4.3ng/uL Strep R-F |
| 6 | 3 | Silk 4.9ng/uL R-strepF |
| 7 | 3 | Silk 5.9 ng/uL R-strepF |
| 8 | Silk without strep tag | 10-07-13 Silk without strep tag 5.1 ng/uL |
| 9 | 2 | 16-07 Silk |
| 10 | 3 | 16-07 Silk |
Sander
did a ligation reaction on silk 1 with two of the Signal Sequences (MotB and EstA) at 1:1 ratio.
ran a gel with 0.8% agarose at 90V for 34 min.
no bands visible but external factors migh be influencinfg imaging zo samples were stored in freezer (box TEMP: marked on top with M-S and E-S on side with lig m-silk and lig e-silk)
