- 2 approaches with psB4A5
- final concentration:

- approach 1: 30 ng/µl
- approach 2: 40 ng/µl

Q5 2X MasterMix: 5 µL
10 uM Primer: each 0,5 µL
Template: 1 µL of 1:100 diluted Miniprep (about 0,5 ng)
ddH2O: 3 µL
total: 10 µL

- let it run on gel for purification
- 140 V a 40 min
- cut out was done by Natalie and Pauline

- made new PCR using Q15/Q16 primers and psB4A5 plasmid fpr linearization
- used 3 different DNA concentrations and 3 different annealing temperatures (9 reactions total)

component	1 reaction: volume in µl	10 reaction MM: volume in µl
HiQ	5	50
primer	1,25	12,5
DNA	variable (1 µl)	variable
H2O	17,5	175
final volume	25	250

- annealing temperatures: A = 60°C/B = 64,5°C/C = 68°C
- DNA concentrations: 0,1 = 1:1000 / 1 = 1:100 / 10 = 1:10
Scheme:

0,1 A	0,1 B	0,1 C
1 A	1 B	1 C
10 A	10 B	10 C

Wednesday, 08/16/17

1. Gel run of psB4A5 gradient PCR (A,B,C,0.1,1,10)
2. Gel purification of dCAs9 A + B fragments and 1A-psB4A5 linearized plasmid
3. Fusion PCR of dCAs9 fragments A + B

component	volume in µl
Q5-MM	12,5
DNA	2(1 pro fragment)
H2O	10,5
final volume	25

IMPORTANT: Fusion PCR done like team munich!
https://static.igem.org/mediawiki/2014/c/c9/LMU_Munich14_FusionPCR.pdf


Thursday, 08/17/17

1. dCas9:

pSB4A5: DpnI-digestion: 1 µl added to the PCR mix and incubated 1 h at 37°C SLiCE reaction of pSB4A5 with IAAM-MS2 + IAAH-PP7: because of to low DNA concentration we needed to increase the concentration by drying the DNA for this reaction we take: 2,8 µl backbone (50 ng, 3395 bp, 18 ng/µl) + 32,2 µl IAAM-MS2 (1:10 ratio, 2188 bp, 10 ng/µl) + 26,4 µl IAAH-PP7 (1:10 ratio, 1790 bp, 10 ng/µl) all in one eppi + 38,6 µl ddwater to get 100 µl to the 100 µl we added: + 1/10 of original volume (100µl) 3 M Sodium acetate, pH 5,2 --> 10 µl + 2 x original volume 100% ethanol (needs to be cooled down for 20 min at -20°C) --> 200 µl mix thorougly put 30 min to -20°C (in this time cool down centrifuge to 4°C) centrifuge 10 min with maiximal speed at 4°C discard supernatant with a pipette, BE CAREFUL!!!, pellet is maybe (in most cases, because we don't have much DNA) nit visible, pay attenteion on the orinetation of the Eppis in the centrifuge (lids to the outside) and go with the pipette onto this side of the Eppi, where the pellet DON'T is, DON'T TOUCH THE PELLET!!! add 300 µl 70% ethanol (needs to be cooled down for at least 20 min at -20°C) centrifuge 10 min with maiximal speed at 4°C again CAREFULLY discard supernatant with a pipette make a short spin 10-30 sec (Short-Button) and take carefully additional supernatant with a pipette put Eppi open lid for 10 min to 37°C add SLiCE reaction directly to this Eppi (NO RESUSPENSION BEFORE) SLiCE reaction: Eppi with dried backbone, IAAM-MS2 and IAAH-PP7 + 1 µl 10 x SLiCE buffer + 1 µl PPY SLiCE extract + 8 µl ddwater --> 10 µl SLiCE reaction incubate 1 h at 37°C, no shaking (flip a few times to the Eppi to resuspend the DNA) Transformation in E.coli clls with all the 10 µl SLiCE reaction in the same Eppi like the transformation protocol, no changes from protocol 200 µl to Ampicillin-LB-agar plates and centrifuge rest of the culture, discard most of the supernatant, resuspend in the rest supernatant and put this resuspenden rest also to an Ampicillin-LB-agar plate plates over night to 37°C for tomorrow: Colony-PCR with the SLiCE plate and at the same time preparation of the Slakowski-Assay plate