Team:Groningen/Labwork/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. The second round of PCR gave nice bands around the expected 900 bp. Therefore a gel extraction PCR clean up is done. 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 digestion 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	BBa_k818000 from
1	4 mL LB, 8 uL Amp	iGEM 2012
2	4 mL LB, 8 uL Amp	iGEM 2012
3	4 mL, LB 8 uL Amp (negative control)	iGEM 2012
4	4 mL LB, 8 uL Amp, 4 uL IPTG	iGEM 2012
5	4 mL LB, 8 uL Amp, 4 uL IPTG	iGEM 2012
6	4 mL, 8uL Amp	iGEM 2013
7	4 mL, 8 uL Amp, 8uL IPTG	iGEM 2013
8	4 mL, 8 uL Amp (negative control)	iGEM 2013

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.

Sander and Mirjam

did a gel purification of the Silk 2 and 3 PCR product.
samples were stored in freezer (box TEMP: marked on top with 2,1; 2,2; 2,3; 3,1; 3,2 and 3,3. on side with gel pur silk [top])

Claudio

4 colonies (supposed to contain the new backbone derived from Munich) are picked from the plate and inoculated in LB + Ampicillin.