"
Team:Groningen/Labwork/22 July 2013
From 2013.igem.org
| Line 80: | Line 80: | ||
<br>Inoculated the BBa_k818000 backbone again, because the previous one was suspected of being white instead of red. | <br>Inoculated the BBa_k818000 backbone again, because the previous one was suspected of being white instead of red. | ||
| - | <br>Also IPTG was added to some of the samples to see its effects. | + | <br>Also IPTG was added to some of the samples to see its effects. Cells are to grow overnight at 37 C. |
<table border = "1"> | <table border = "1"> | ||
Revision as of 14:39, 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. Although no ligation product is found, a PCR reaction is made to obtain higher concentrations. The annealing temperature for MotB, FliZ and LytB is 70 degrees Celsius. For EstA 77 is used. The fragments will be around 1000 bp.
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 |
Inoculated the BBa_k818000 backbone again, because the previous one was suspected of being white instead of red.
Also IPTG was added to some of the samples to see its effects. Cells are to grow overnight at 37 C.
| Sample | Composition |
| 1 | 4 mL LB, 8 uL Amp |
| 2 | 4 mL LB, 8 uL Amp |
| 3 | 4 mL, LB 8 uL Amp (negative control) |
| 4 | 4 mL LB, 8 uL Amp, 4 uL IPTG |
| 5 | 4 mL LB, 8 uL Amp, 4 uL IPTG |
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 migth be influencing imaging so 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)
Run a gel with the new silk 2 an 3 PCR products to determine if the silk is present. 1.5% agarose 90V 45min.
