Team:Marburg/Labbook/InterLab
15/04/22
Primer Design
Primer Design iILS1-915/04/26
Miniprep
15/06/03
PCR
PCR Mix | PCR1 | PCR2 | PCR3 | PCR4 | PCR5 | PCR6 |
0,5ul | BB1 3108 | GFP1 3504 | BB2 3109 | GFP2 3504 | BB3 111 | GFP3 3504 |
1ul | ILS2 | ILS4 | ILS2 | ILS7 | ILS2 | ILS9 |
1ul | ILS3 | ILS5 | ILS6 | ILS5 | ILS8 | ILS5 |
31,5ul | H2O | H2O | H2O | H2O | H2O | H2O |
10ul | Buffer | Buffer | Buffer | Buffer | Buffer | Buffer |
4ul | dNTPs | dNTPs | dNTPs | dNTPs | dNTPs | dNTPs |
2ul | GXL Polymerase | GXL Polymerase | GXL Polymerase | GXL Polymerase | GXL Polymerase | GXL Polymerase |
Cycler Protocol (30x) | PCR1,3,5 | PCR2,4,6 |
98°C | 10s | 10s |
55°C | 15s | 15s |
68°C | 22s | 10s |
end cycle | ||
10°C | store | store |
PCR
Repeat PCR of BB1, BB2Comments: unsatisfied yield for PCR1, PCR3 -> pcr of pcr (3', 4') and repeat PCR with original template (1',2')
Gel extraction
Gel extraction of PCR2,4,615/06/04
PCR
PCR of BB111 (BB3)15/06/08
Gel electrophoresis
PCR1'-4'PCR
PCR of GFP (PCR2,4,6)Gel electrophoresis
PCR2,4,615/06/09
PCR
Repeating PCR of PCR1, PCR3, PCR4Gel extraction
Gel of PCR1,3,4DpnI digest
Add 2ul of DpnI to the PCR, incubate for 30min @37°CGibson Assembly
Gibson1: BB1+GFP1= pILS1Gibson3: BB3+GFP3= pILS3
Gibson 1 | Gibson 2 | |
BB | 1,4ul | 1,5ul |
GFP | 3,23ul | 2,6ul |
H2OC | 5,4ul | 5,9ul |
Gibson MM | 10ul | 10ul |
Transformation
Transform 2ul Gibson mix of Gibson 1 and Gibson3 into NEB electrocompetent cellsRecover 1h, plate 100ul
15/06/11
CPEC1
CPEC 1 | CPEC 3 | |
BB | 3,34ul | 3,78ul |
GFP | 0,69ul | 0,56ul |
H2OC | 5,97ul | 5,65ul |
Analytical digest
BB2 | 4ul |
H2O | 3ul |
XbaI | 1ul |
SacI | 1ul | Cutsmart | 1ul |
30min @37°C
Gel electrophoresis
Check the digest of BB215/06/12
Analytical digest of BB2
Digest 1 | Digest 2 | Digest 3 | |||
BB2 | 4ul | BB2 | 7ul | BB2 | 4ul |
H2O | 3ul | H2O | 2ul | ||
PvuII | 1ul | EcoRI | 1ul | EcoRI | 1ul | PstI | 1ul | PstI | 1ul | Cutsmart | 1ul | Cutsmart | 1ul | Cutsmart | 1ul |
Incubate 30min @37°C
Gel elxtraction
Check the different digests of BB215/06/13
Transformation
Transform following plasmids into NEB Turbo (Chemical competent):K823005 Promoter
K823008 Promoter
K823013 Promoter
Gibson 1
Gibson3
CPEC1
CPEC3
15/06/15
Inoculation
K823005, K823008 & K823013 picked & inoculated into LB-mediumIncubated @ 37 °C, 250 rpm
Miniprep
Prep the Colonies of the ONFrom now on:
K823005: BB04
K823008: BB05
K823013
15/06/17
PCR
PCR9 | PCR10 | |
0,5ul template | BB K8230008 | GFP4 (from PCR6) |
1ul Primer | iILS8 | iILS9 |
1ul Primer | iILS2 | iILS5 | 10ul | buffer | buffer | 4ul | dNTPs | dNTPS | 2ul | Polymerase | Polymerase | 31,5ul | H2O | H2O |
Gel electrophoresis
Gel of Digest of the 06/16Lane1: GFP04, Lane 2: BB08
Gelextraction
Extraction of BB08 and GFP0415/06/18
CPEC
CPEC2 (pILS5)CPEC2 | |
2,5ul | BB08 |
4,29ul | GFP04 |
27,21 | H2O | 10ul | buffer | 4ul | dNTPs | 2ul | Polymerase |
15/06/24
PCR
PCR for pILS4 and pILS6PCR Mix | PCR7 | PCR8 | PCR11 | PCR12 |
1ul | BB05 | GFP | BB13 | GFP |
1ul | Primer iILS2 | Primer iILS5 | Primer iILS18 | Primer iILS5 |
1ul | Primer iILS16 | Primer iILS15 | Primer iILS115 | Primer iILS17 | 31ul | H2O | H2O | H2O | H2O | 10ul | buffer | buffer | buffer | buffer | 4ul | dNTPS | dNTPS | dNTPS | dNTPS | 2ul | Polymerase | Polymerase | Polymerase | Polymerase |
Gel electrophoresis
Load the PCR on a gel to cut outGel extraction
Extract all PCRs15/06/29
CPEC
pILS4 | pILS6 | ||
BB04 | 0,2ul | BB06 | 0,8ul |
GFP4 | 1,2ul | GFP6 | 0,9ul |
H2O | 32,62ul | H2O | 32,35ul | buffer | 10ul | buffer | 10ul | dNTPs | 4ul | dNTPs | 4ul | Polymerase | 4ul | Polymerase | 4ul |
Next day: Transformation - no colonies grew
15/07/01
PCR
PCR for pILS5PCR Mix | PCR9 | PCR10 |
0,5ul | BB08 | GFP |
1ul | iILS8 | iILS9 |
1ul | iILS2 | iILS5 | 31,5ul | H2O | H2O | 10ul | buffer | buffer | 4ul | dNTPs | dNTPs | 2ul | Polymerase | Polymerase |
PCR
PCR of GFP4, GFP6, BB4, BB6Gel showes that BB6 was not succesful
Gel extraction
extract BB4, GFP4, GFP6 from gelPCR
Repeat PCR of BB615/07/02
CPEC5, CPEC4
CPEC5 | CPEC4 | ||
BB4 | 3,5ul | BB04 | 1,9ul |
GFP5 | 5,1ul | GFP4 | 2,2ul |
H2O | 25,4ul | H2O | 29,9ul | buffer | 10ul | buffer | 10ul | dNTPs | 4ul | dNTPs | 4ul | Polymerase | 4ul | Polymerase | 4ul |
Transformation
Transform CPECS into NEB (chemically)no colonies grew
15/07/06
CPEC5, CPEC4
CPEC5 | CPEC4 | ||
BB4 | 3,5ul | BB04 | 1,9ul |
GFP5 | 5,1ul | GFP4 | 2,2ul |
H2O | 25,4ul | H2O | 29,9ul | buffer | 10ul | buffer | 10ul | dNTPs | 4ul | dNTPs | 4ul | Polymerase | 4ul | Polymerase | 4ul |
use different cycler protocol
15/07/07
Transformation
Transform CPEC4, 5 into NEB turbo (chemical transformation)Glycerol stocks
Prepare glycerol stocks (siGEM08, -siGEM11) of pILS4,5,6 of overnight culturesMiniprep
Miniprep of Overnight CulturesPCR
Repeat PCR7-1215/07/08
PCR
PCR of 7-12PCR7,8,9,10,12 were successful
Repeat PCR11
Gel extraction
Extract PCRS that workedCPEC
CPEC4 | CPEC6 | ||
BB4 | 0,41ul | BB06 | 0,37ul |
GFP4 | 4,23ul | GFP6 | 9,26ul |
H2O | 29,35ul | H2O | 24,37ul | buffer | 10ul | buffer | 10ul | dNTPs | 4ul | dNTPs | 4ul | Polymerase | 4ul | Polymerase | 4ul |
Transformation
Transform PEC5 and CPEC6 of the 6th into NEB chem comp. Use 5ul of DNA.No colonies for CPEC4 the next day
Colonies for CPEC5.
Inoculation
Inoculate 8 colonies of CPEC5 in 5ml LB-CAM and incubate @37°C for 6h.Minirep
Prep the cultures.Analytical digest
Digest the 8 plasmids with PvuII15/07/10
Gel extraction
Extract PCR BB6, BB4, GFP4 and GFP6 out of GelCPEC
CPEC4 | CPEC6 | ||
BB4 | 1,25ul | BB06 | 8ul |
GFP4 | 0,42ul | GFP6 | 0,42ul |
H2O | 32,33ul | H2O | 31,58ul | buffer | 10ul | buffer | 10ul | dNTPs | 4ul | dNTPs | 4ul | Polymerase | 4ul | Polymerase | 4ul |
PCR
New PCR of BB6Gel extraction
Extract BB6 BB6CPEC
CPEC4 | CPEC6 | ||
BB4 | 2,4ul | BB06 | 1,3ul |
GFP4 | 0,7ul | GFP6 | 0,8ul |
H2O | 30,9ul | H2O | 31,9ul | buffer | 10ul | buffer | 10ul | dNTPs | 4ul | dNTPs | 4ul | Polymerase | 4ul | Polymerase | 4ul |
15/07/11
Transformation
Transform all previous CPECs into NEB turbo, chemical competent cells15/07/12
Inoculation
Pick 8 colonies of each construct and grow them in LB CAM15/07/13
Miniprep
Prep all the colonies from construct 4 and 6Analytical digest
Digest with PvuIITEXT
Text
lalala15/09/02
Transformation
Trafo of constr. 10,4+11,4+12,4 in NEB TurboPCR
PCR of P30+31PCR Cleanup
PCR-Clean up of P30+31CPEC
CPEC of construct 5TEXT
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