Team:CU-Boulder/Test

From 2014.igem.org

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CRISPR/Cas9 Technology

Find out about the modern ways of Killing Tuberculosis using the CRISPR/Cas technology!

DAY NOTES

ASDF

Saturday 13^th July

PCR circular and linear, Conjugation of XL-10 (with sSP011), Patches, Digestion M13 backbone, RFP insert, Gel of PCR

PCR circular and linear, 40.5°C, 60° of pSB1A3, pSB1C3

Reagent	Volume
	1x
Nuclease-free water	37.25 ul
5x Phusion HF Buffer	10 ul
10 mM dNTPs	1 ul
Forward Primer (10 uM)	0.5 ul
Reverse Primer (10 uM)	0.5 ul
Template Plasmid	0.25 ul
Phusion DNA Polymerase	0.5 ul
Total Volume	50 ul

Thermocycler Protocol: NEB Phusion
	Temp	Time
Start	98°C	30 sec	Melt

Cycle 1	98°C	5 sec	Melt	35 cycles
Cycle 2	40.5°C / 60°C	25 sec	Anneal
Cycle 3	72°C	5 min	Extend

Finish	72°C	5 min	Extend
Store	10°C	Forever	Store

Conjugation of XL-10 (with sSP011)
1) From O/N cultures Dilute strains 1/100 in LB
2) Wait for OD to reach O,2
3) Prepare tube (in BD tubes) :
- Tube = 0,5mL LB with Strain (sSP011) + 0,5mL LB with Strain (XL-10 Kan)
4) Incubate 2 hours at 37°C (actually not in the shaker, but we accidently kept them in the shaker...)
5) Plate 20ul for mixed tube on LB antiobiotics (Tet, Kan)
6) Incubate overnight at 37°C

Patches
check and make new ones

Digestion M13 backbone, RFP insert
for Backbone (M13mp18 plasmid): 3ug
7,58 ul plasmid (c=395ng/ul)
3 ul EcoRI
3 ul PstI
3ul 10x Fast Digest
13,42 ul H20
incubate for 12 min on 37°
heat inactivation: 80° 5 min
for insert (BBa_J04450): 5ug
31,4 ul plasmid
5 ul EcoRi
5 ul PstI
3,6 ul H20
incubate for 20 min at 37°
heat inactivation: 80° 5min

Gel of PCR (Amp 40.5 circ, lin, Chl 60° lin, circ)
100V, 20 min 1% gel

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Team:CU-Boulder/Test

From 2014.igem.org

Saturday 13th July

Saturday 13^th July