"
Team:Marburg SYNMIKRO/Notebook
From 2012.igem.org
Contents |
Week 1
09.07. - 15.07.2012
09.07.2012
- Inoculation
- E. coli strain C600 Mucts 62' in DYT-medium
10.07.2012
- Isolation of chromosomal DNA
- E. coli strain C600 Mucts 62
- Inoculation
- E. coli strain DH5a and TOP10 in DYT-medium
11.07.2012
- Preparation of chemically competent E. coli cells
- TOP10
12.07.2012
- Transformation
- GFP, GFP_fusion, CFP, mRFP, RBS, P108, P100, PLac with E.coli Top10
| Name | Biobrick | Plate | Well | Backbone | Resistance |
|---|---|---|---|---|---|
| GFP | [http://partsregistry.org/Part:BBa_E0040 BBa_E0040] | 1 | 14K | pSB1A2 | Ampicillin |
| GFP_fusion | [http://partsregistry.org/Part:BBa_K125500 BBa_K125500] | 3 | 2P | pSB1A2 | Ampicillin |
| CFP | [http://partsregistry.org/Part:BBa_E0020 BBa_E0020] | 1 | 6A | pSB1A2 | Ampicillin |
| mRFP | [http://partsregistry.org/Part:BBa_E1010 BBa_E1010] | 1 | 18F | pSB2K3 | Kanamycin |
| RBS | [http://partsregistry.org/Part:BBa_J61100 BBa_J61100] | 1 | 5J | pSB1A2 | Ampicillin |
| P108 | [http://partsregistry.org/Part:BBa_J23108 BBa_J23108] | 2 | 2E | BBa_J61002 | Ampicillin |
| P100 | [http://partsregistry.org/Part:BBa_J23100 BBa_J23100] | 1 | 18C | BBa_J61002 | Ampicillin |
| PLac | [http://partsregistry.org/Part:BBa_I14032 BBa_I14032] | 2 | 11P | pSB2K3 | Kanamycin |
Week 2
16.07. - 22.07.2012
16.07.2012
- PCR
- AmiC (primer: MS1 + MS2)
- HU (primer: MS3 + MS4)
- GroES (primer: MS5 + MS6)
- GixR (primer: MS7 + MS8)
- Enhancer (primer: MS9 + MS10)
- ⇒ template-DNA: chromosomal DNA, annealing: 30 sec; 60 °C, elongation: 30 sec; 72 °C, 35 cycles
- Inoculation
- GFP
- GFP_fusion
- CFP
- mRFP
- RBS
- P108
- LacIQ
- P100
17.07.2012
- Plasmid preparation
- GFP
- GFP_fusion
- CFP
- mRFP
- RBS
- P108
LacIQ- P100
18.07.2012
- Agarose gel
- pipet scheme
Marker|enhancer|GroES|AmiC|HU|GixR|GFP|GFP-Fusion|CFP|mRFP|RBS|LacIQ|J23108|J23100|λ-DNA (bild 1)
- DNA Gel extraction
- Enhancer
- GroES
- HU
- GixR Hybridization
- 5 µl primer MS7 (100 µM) + 5 µl primer MS8 (100 µM)
- 5 min 99°C
- over night, room temperature
- -80°C
19.07.2012
- Fill-in reaction (primer extension)
- Enhancer
- Restriction
- Enhancer (EcoRI/PstI)
- PCR
- AmiC (primer: MS1 + MS2)
- ⇒ template-DNA: chromosomal DNA, annealing: 30 sec; 85 °C, elongation: 36 sec; 72 °C, 35 cycles, 1) 2 µl DMSO; 1 µl chrom. DNA | 2) 2 µl DMSO; 5 µl chrom. DNA | 3) without DMSO; 1 µl chrom. DNA | 4) without DMSO; 5 µl chrom. DNA | 5) 2 µl DMSO; without chrom. DNA
- Test restriction
- 7) CFP (EcoRI/PstI)
- 8) GFP (EcoRI/PstI)
- 9) GFP-fusion (EcoRI/PstI)
- 10) mRFP (EcoRI/PstI)
- ⇒ all negative
- Agarose gel
- pipet scheme
1)|2)|3)|4)|5)|marker|7)|8)|9)|10) (bild 2)
- Control digest
- CFP (EcoRI/PstI)
- GFP (EcoRI/PstI)
- GFP-fusion (EcoRI/PstI)
- mRFP (EcoRI/PstI)
- ⇒ restriction over night
20.07.2012
- Agarose gel
- pipet scheme
- ⇒ marker|GFP|GFP-fusion|CFP|mRFP (all positive)
(bild 3)
Week 3
23.07. - 29.07.2012
26.07.2012
- Restriction
- pSB1T3 (XbaI/SpeI and DpnI)
- pSB1A3 (XbaI/SpeI and DpnI)
- pSB1K3 (XbaI/SpeI and DpnI)
- pSB1C3 (XbaI/SpeI and DpnI)
- Ligation
- GixR + pSB1A3
- Enhancer + pSB1K3
- cyclisation of pSB1C3, pSB1K3, pSB1A3, pSB1T3 (couldn’t work)
Week 4
30.07. - 05.08.2012
30.07.2012
- PCR
- CFP (primer: MS16 + MS17) ⇒ template-DNA: BBa_E0010
- ⇒ positive
- mRFP (primer: MS14 + MS15) ⇒ template-DNA: BBa_E1010
- ⇒ positive
- Gin (primer: MS11 + MS13) ⇒ template-DNA: chromosomal DNA (that was wrong)
- RBS-Gin (primer: MS13 + MS12) ⇒ template-DNA: chromosomal DNA (that was wrong)
- ⇒ annealing: 30 sec; 60 °C, elongation: 40 sec; 72 °C, 35 cycles
- Transformation
- all vector-backbone-ligations
- Enhancer (K3)
- GixR (A3)
31.07.2012
- Result
- transformation
- pSB1A3: 3 colonies
- pSB1K3: 60 colonies
- pSB1C3: 9 colonies
- pSB1T3: 0 colonies
- Enhancer (K3): 0 colonies
- GixR (A3): 0 colonies
- Agarose gel
- pipet scheme
- ⇒ gix|marker|AmiC from 16.07.| AmiC from 16.07.| AmiC from 19.07.| AmiC from 19.07.
- ⇒ all negative
(bild 4,5)
- Agarose gel
- pipet scheme
- ⇒ marker|RBS-Gin from 30.07.|AmiC from 16.07.|Gin from 30.07.|CFP from 30.07.|mRFP from 30.07.|control
- ⇒ all negative
- Resuspension of a biobrick
- BBa_B0010 (terminator)
- DNA Gel extraction
- CFP
- mRFP
- PCR
- AmiC (primer: MS1 + MS2)
- RBS-Gin (primer: MS12 + MS13)
- Gin (primer: MS11 + MS13)
- ⇒ template-DNA: chromosomal DNA, annealing: 30 sec; 55 °C, elongation: 40 sec; 72 °C, 35 cycles
- Restriction
- pSB1A3 (EcoRI/PstI)
- CFP (EcoRI/PstI)
- mRFP (EcoRI/PstI)
- Transformation
- pSB1A3
- pSB1C3
- pSB1T3
- Terminator
- GixR
- Enhancer
- Inoculation
- pSB1A3
- pSB1C3
- pSB1K3
- Ligation
- pSB1A3 + CFP
- pSB1A3 + mRFP
- GixR Hybridization
- 5 µl primer MS7 (100 µM) + 5 µl primer MS8 (100 µM)
- 5 min 99°C
- over night, room temperature
- -80°C
- DNA Gel extraction
- AmiC
- Ligation
- AmiC + pSV2 (suicide vector)
01.08.2012
- Result
- transformation
- Only BBa_B0010 colonies present
- Plasmid preparation
- pSB1A3 ⇒ only clone 1, 3 Positive
- pSB1C3 ⇒ all negative
- pSB1K3 ⇒ all negative
- Ligation
- GixR hyb. + pJET2_1
- ⇒ over blunt ends
- Restriction
- GixR hyb. (EcoRI/PstI)
- pSB1C3 (EcoRI/PstI)
- Agarose gel
- pipet scheme
- Marker|RBS-Gin1 +DMSO|RBS-Gin2 -DMSO|Gin1 +DMSO|Gin2 -DMSO|control
(bild 6)
- Agarose gel
- pipet scheme
- Marker|AmiC1 +DMSO| AmiC2 –DMSO
(bild 7)
- DNA Gel extraction
- RBS-Gin
- Gin
- AmiC
- Test restriction
- pSB1A3 (EcoRI)
- Restriction
- RBS-Gin (EcoRI/PstI)
- Gin (EcoRI/PstI)
- AmiC (EcoRI/PstI)
- pSB1C3 (EcoRI/PstI)
- pSB1K3 (EcoRI/PstI)
- pSB1T3 (EcoRI/PstI)
- Ligation
- cyclisation of pSB1T3
- Fill-in reaction (primer extension)
- Enhancer
- Ligation
- Enhancer and GixR in pSV2
- Enhancer in pSB1K3
- GixR in pSB1C3
- Agarose gel
- pipet scheme
- Marker| pSB1A3 1,2,3| pSB1C3 1,2,3,4| pSB1K3 1,2,3,4
(bild 8)
- pSB1A3 c2
- pSB1C3 c4
- pSB1K3 c3
- Transformation
- pSB1A3 religation
- CFP in A3
- mRFP in A3
- AmiC in pJET
- GixR in pJET
- Enhancer in pJET
- GixR in C3
- Enhancer in K3
- Ligation
- AmiC + pSB1T3
- RBS-Gin + pSB1K3
- Gin + pSB1K3
02.08.2012
- Resuspension of a biobrick
- pSB3C5
- ⇒ for ccdB
- Plasmid preparation
- BBa_B0010 (terminator)
- Test restriction
- Terminator c1,2,3,4 (EcoRI/PstI)
- Agarose gel
- pipet scheme
- Marker|Terminator 1,2,3,4
(bild 9)
- Transformation
- pSB1T3 recycled
- AmiC in T3
- Gin-RBS in K3
- Gin in K3
- pSB3C5 linear (false)
03.08.2012
- Plasmid preparation
- Enhancer in pJET
Week 5
06.08. - 12.08.2012
06.08.2012
- Resuspension and transformation of a biobrick
- BBa_J04450 in pSB1A3
- BBa_J04450 in pSB1C3
- BBa_J04450 in pSB1K3
- BBa_J04450 in pSB1T3
- Restriction
- GFP-fusion (EcoRI/SpeI)
- Terminator (XbaI/PstI)
- Promoter BBa_J23100 (EcoRI/SpeI)
- Promoter BBa_J23108 (EcoRI/SpeI)
- RBS (XbaI/PstI)
- Ligation
- RBS-Gin in T3
- Gin in T3
- GFP-fusion (EcoRI/SpeI) + Terminator (XbaI/PstI) in pSB1K3
- Promoter BBa_J23108 (EcoRI/SpeI) + RBS (XbaI/PstI) in pSB1K3
- Promoter BBa_J23100 (EcoRI/SpeI) + RBS (XbaI/PstI) in pSB1K3
- GixR Hybridization
- 1(3) µl primer MS7 (100 µM) + 1(3) µl primer MS8 (100 µM) + T4-Ligasebuffer
- 5 min 95°C
- over night, room temperature
- -80°C
- Transformation
- GixR in C3
- Gin in T3
- RBS-Gin in T3
- mRFP in A3
- CFP in A3
07.08.2012
- Transformation
- GFP-fusion
- CFP
- J23108-RBS
- J23100-RBS
- Plasmid preparation
- GixR 1,2,3
- Restriction
- GixR hyb. 1,2,3 (EcoRI/PstI)
- pSB1C3 (EcoRI/PstI)
- Ligation
- GixR hyb. 1,2,3 in pJET
- GixR hyb. 1,2,3 in C3
- Transformation
- GixR hyb. 1,2,3 in pJET
- GixR hyb. 1,2,3 in C3
- Test restriction
- pSB3C5 (XbaI)
- pJET (XbaI)
- Agarose gel
- pipet scheme
- Marker|pSB3C5 c1a,2a,3a,4a|Marker|pSB3C5 c1b,2b,3b,4b|Marker|Enhancer-pJET c1,2,3,4|Marker
(bild 10)
- ⇒ positive: pSB3C5 c3a,4a,3b,4b,; Enhancer-pJET c1,2,3,4
08.08.2012
- Result transformation
- GFP-fusion (20 clones)
- J23108-RBS (20 clones)
- J23100-RBS (20 clones)
- GixR hyb. 1,2,3 in pJET (10 clones)
- GixR hyb. 1,2,3 in C3 (10 clones)
- CFP (5 clones)
- Restriktion
- Enhancer in pJET (EcoRI/PstI)
- pSB1C3 (EcoRI/PstI)
- Agarose gel
- pipet scheme
- Marker| Enhancer|pSB1C3
(bild 11)
- DNA Gel extraction
- Enhancer
- pSB1C3
- Ligation
- Enhancer 1,2 in C3
- Transformation
- Enhancer 1,2 in C3
- C3
- mRFP
09.08.2012
- Result transformation
- GFP-fusion ⇒ positive
- J23108-RBS ⇒ positive
- J23100-RBS ⇒ positive
- CFP ⇒ positive
- GixR in C3 c1 ⇒ negative
- GixR in C3 c2 ⇒ negative
- GixR in C3 c3 ⇒ negative
- Plasmid preparation
- GFP-fusion
- J23108-RBS
- J23100-RBS
- CFP
- Test restriction
- GFP-fusion (EcoRI/PstI)
- J23108-RBS (EcoRI)
- J23100-RBS (EcoRI)
- CFP (EcoRI/PstI)
- Agarose gel
- pipet scheme
- Marker|GFP-fusion 1,2,3,4,5,6,7,8,9| Marker|10,11,12,13,14,15,16,17,18
(bild 12)
- Marker|J23108-RBS 1,2,3,4,5,6,7,8,9| Marker|10,11,12,13,14,15,16,17,18
(bild 13)
- Marker|J23100-RBS 1,2,3,4,5,6,7,8,9| Marker|10,11,12,13,14,15,16,17,18
(bild 14)
- Marker|GFP-fusion 19,20| J23108-RBS 19,20| J23100-RBS 19,20| Marker |CFP 1,2,3,4,5
(bild 15)
- Inoculation
- GixR in C3
- Gin in T3
- Gin-RBS in T3
- pSB1C3
- Enhancer in C3
- mRFP in A3
Week 6
13.08.2012 - 19.08.2012
13.08.12
- PCR
- GroES (primer: MS5 + MS6 (b))
- HU (primer: MS3 + MS4 (b))
- ⇒ with and without DMSO, template-DNA: chromosomal DNA, annealing: 30 sec; 60 °C, elongation: 40 sec; 72 °C, 35 cycles
- Agarose gel
- pipet scheme
- Marker|HU +DMSO|HU -DMSO|GroES +DMSO|GroES –DMSO|control
(bild 16)
- Transformation
- LacIQ (BBa_I14032)
14.08.12
- Restriction
- pSB1K3 (EcoRI/PstI)
- Enhancer 3,4 (EcoRI/ PstI)
- GFP-fusion (EcoRI/SpeI)
- Terminator (XbaI/PstI)
- Test restriction
- RBS-Gin in T3 (EcoRI/ PstI)
- Ligation
- GFP-fusion (EcoRI/SpeI) + Terminator (XbaI/PstI) in pSB1K3 (EcoRI/PstI)
- Promotor J23108 (EcoRI/SpeI) + RBS (XbaI/PstI) in pSB1K3 (EcoRI/PstI)
- Enhancer 3,4 (EcoRI/ PstI) + pSB1K3 (EcoRI/PstI)
- Transformation
- GFP-fusion-Terminator in pSB1K3
- Promotor-RBS in pSB1K3
- Enhancer 3 in pSB1K3
- Enhancer 4 in pSB1K3
- GixR in pJET
15.08.2012
- PCR
- GroES (primer: MS5 + MS6 (b))
- HU (primer: MS3 + MS4 (b))
- ⇒ with and without DMSO, template-DNA: chromosomal DNA, annealing: 30 sec; 55 °C, elongation: 30 sec; 72 °C or annealing/elongation: 30 sec; 72 °C, 35 cycles
- Agarose gel
- pipet scheme
- Marker|HU +DMSO|HU -DMSO|GroES +DMSO|GroES –DMSO| HU +DMSO two-step PCR|HU -DMSO two-step PCR |GroES +DMSO two-step PCR |GroES –DMSO two-step PCR
(bild 17)
- Resuspension of a biobrick
- BBa_J04450 in pSB1A3
- BBa_J04450 in pSB1T3
- BBa_J04450 in pSB1C3
- BBa_J04450 in pSB1K3
- Agarose gel
- pipet scheme
- Marker|HU -DMSO|HU +DMSO|GroES -DMSO|GroES +DMSO
(bild 18)
- ⇒ 2-step PCR didn’t work, HU 1,2
- DNA Gel extraction
- HU 1,2
- Transformation
- BBa_J04450 in pSB1A3
- BBa_J04450 in pSB1T3
- BBa_J04450 in pSB1C3
- BBa_J04450 in pSB1K3
- Test restriction
- pSB1T3 (EcoRI/PstI and DpnI)
- pSB1K3 (EcoRI/PstI and DpnI)
- pSB1C3 (EcoRI/PstI and DpnI)
- pSB1A3 (EcoRI/PstI and DpnI)
16.08.2012
- PCR
- GroES (primer: MS5 + MS6 (b))
- ⇒ with and without DMSO, template-DNA: chromosomal DNA, annealing: 30 sec; 55 °C, elongation: 30 sec; 72 °C , 35 cycles
- Agarose gel
- pipet scheme
- Marker| GroES 1, 2 +DMSO
(bild 19)
- Plasmid preparation
- P-RBS
- Test restriction
- P-RBS (EcoRI/PstI)
- Agarose gel
- pipet scheme
- control|P-RBS|marker
(bild 20)
- Restriction
- Enhancer 3,4 (EcoRI/ PstI)
- HU (EcoRI/ PstI)
- AmiC (EcoRI/ PstI)
- Promoter, GFP (EcoRI/SpeI)
- RBS; Terminator (XbaI/PstI)
- Ligation
- HU, AmiC in pSB1A3
- Enhancer 3,4 in pSB1K3
- P-RBS in pSB1K3
- GFP fusion-Terminator in pSB1K3
- DNA Gel extraction
- GroES 1, P-RBS
- Transformation
- AmiC
- HU
- Enhancer 3,4, 3 alt,4 alt
- Inoculation
- BBa_J04450 in pSB1A3
- BBa_J04450 in pSB1T3
- BBa_J04450 in pSB1C3
- BBa_J04450 in pSB1K3
- AmiC
- P-RBS
17.08.2012
- Restriction
- GroES (EcoRI/SpeI)
- Ligation
- GroES in pSB1C3
- Transformation
- GroES in pSB1C3
- Test restriction
- P-RBS (EcoRI/PstI)
- BBa_J04450 in pSB1A3 (EcoRI/PstI)
- BBa_J04450 in pSB1T3 (EcoRI/PstI)
- BBa_J04450 in pSB1C3 (EcoRI/PstI)
- BBa_J04450 in pSB1K3 (EcoRI/PstI)
- Agarose gel
- pipet scheme
- marker|BBa_J04450 in pSB1A3 1,2,3,4|BBa_J04450 in pSB1K3 1,2,3,4| marker| BBa_J04450 in pSB1C3 1,2,3,4
(bild 21)
- Agarose gel
- pipet scheme
- marker|P-RBS 1,2,3,4| P-RBS 1,2,3,4| marker| BBa_J04450 in pSB1K3 1,2,3,4
(bild 22)
Week 7
20.08.2012 - 26.08.2012
20.08.2012
- PCR
- Backbone (primer MS24 + MS25)
- ⇒ template-DNA: pSB1A3, pSB1T3, pSB1C3 and pSB1K3, annealing: 30 sec; 55 °C, elongation: 30 sec; 72 °C , 35 cycles
- Agarose gel
- pipet scheme
- marker|: pSB1A3|pSB1T3|pSB1C3|pSB1K3
(bild 23)
- Transformation
- BBa_JH023
- Inoculation
- GroES
- P-RBS
- GFP-T
- Enhancer 3,4
- AmiC
- P-RBS alt
- GFP-T alt
21.08.2012
- Results
- colonies of BBa_JH023
- inoculations except AmiC and P-RBS alt successful
- PCR
- pSB1A3-BBa_J04450 (template DNA)
- pSB1T3-BBa_J04450 (template DNA)
- pSB1C3-BBa_J04450 (template DNA)
- pSB1K3-BBa_J04450 (template DNA)
- used primer --> MS24Prefix_rev + MS25Suffix_fwd
- initial denaturation 98°C - 5min
- denaturation: 98°C - 1min; annealing: 55°C - 30s; elongation: 72°C - 2.5min -->repeat 35 times
- 72°C - 5min --> 4°C - ∞
- miniprep
- GroES, Enh 3/4 (+old), P-RBS (+old), GFP-T (+old)
- Test restriction
- of miniprep
- over night, 37°C
- Agarose gel (1%)
- watch pic
- Inoculation
- E.coli BBa_I14032
- over night, 37°C
22.08.2012
- PCR
- CFP
- mRFP
- initial denaturation 98°C - 5min
- denaturation: 98°C - 10s; annealing: 60°C - 30s; elongation: 72°C - 40s -->repeat 35 times
- 72°C - 5min --> 4°C - ∞
- Agarose gel (1%)
- watch picture
- miniprep
- of LacIQ
- Test restriction
- of LacIQ with EcoRI and PstI
- watch picture
- Agarose gel
- test restriction of GroES and GFP-T (->21.08.12), gel 1%
- watch picture
- test restriction of Enhancer (->21.08.12), gel 2%
- watch picture
- test restriction of Enhancer and P-RBS (->21.08.12), gel 2%
- watch picture
23.08.2012
Svetti please check! -->lacIQ evtl ganz rausnehmen?! ;)
24.08.2012
Svetti please check!
Week 8
27.08.2012 - 02.09.2012
27.08.2012
- Results
- CFP: 1 colony
- CFP (neu): Colonies
- mRFP: colonies
- mRFP (neu): no colonies
- PCR
- template: backbones (pSB1K3/T3/C3/A3
- parameters: 98°C 5min, >> 98°C 1min, 70°C 30s, 72°C 2.5min << x30, 72°C 5min, 4°C ∞
- colony-PCR
- parameters: 98°C 5min, >> 98°C 1min, 72°C 1min << x30, 72°C 5min, 4°C ∞
- Agarose gel of backbone PCR
- no result
- elution of the backbones of Friday the 24th
- Agarose gel of colony PCR
- watch picture
- Inoculation
- pSB1C3_...
- mRFP clone 20,24
- CFP clone 14,15,17,18
- gix clone 1,2
- 5ml LB, over night, 37°C
28.08.2012
- production of competent TOP10
- PCR
- Enhancer, Terminator, Gix, P-RBS
- parameters: 98°C 5', 98°C 1', 66°C 30", 72°C 30", 72°C 5', 4°C ~
- watch picture
- Transformation
- pBad, araC, pSacB
- over night, 37°C
- miniprep
- GixR (1,2)
- CFP (14,15,17,18)
- mRFP (20,24)
- alle in pSB1C3
29.08.2012
- Results
- Transformation: no colonies
- PCR
- Terminator, Gix, Enhancer, P-RBS
- diluted templates: Terminator, P-RBS, Gix 1 --> 1:10
- Enhancer, Gix 2 --> 1:20
- watch picture
- 3AA
- CFP, mRFP with Terminator
- 2µl DNA
- 1µl SpeI
- 2µl Tango
- 15µl dH2O
- --> 1h at 37°C
- +1µl EcoRI
- +2,5µl Tange
- --> 1h at 37°C
- --> inactivation for 20min at 80°C
- Ligation
- 3AA products in pSB1T3
- 18.5µl H2O
- 2.5 µl Buffer
- 1µl Insert CFP or mRFP (EcoRI + SpeI)
- 1µl Insert Terminator (BBa_B0010) (XbaI + PstI)
- 1µl pSB1T3 (EcoRI + PstI)
- 1µl Ligase
- --> 1h at RT
- Transformation
- pBad --> TOP10 + pSB2K3
- CFP-T, mRFP-T --> TOP10 + pSB1T3
- Sequencing
- bitte mal nachschlagen, die Notizen peil ich nicht ;)
30.08.2012
- Results
- Transformation: no colonies --> testing Trafo with BBa_J04450 A/K/T/C
- 2 colonies at pBad-Trafo from 28.08.2012 --> liquid culture for miniprep
- 1 colony at pSacB-Trafo from 28.08.2012 --> tested on succrose-sensitivity
31.08.2012
- PCR
- SacB
- Parameters No.1: 95°C 5', >>95°C 30", 60°C 30", 72°C 1'<< x30, 72°C 5', 4°C
- Parameters No.2: 95°C 5', >>95°C 30", 72°C 1.5'<< x30, 72°C 5', 4°C
- Agarose gel
- watch picture
- cut out lines at 1.5kb
- Ligation
- 3AA (CFP-T, mRFP-T)
- watch 29.08.
- used vector: pSB1K3/T3
- Transformation
- of ligation in TOP10
- miniprep
- pBad/araC K.1+2
- restriction
- GixR rehybridization
- cut with EcoRI and PstI
- ligation
- 1µl GixRhyb E+P
- 1µl pSB1C3 E+P
- 1µl Th-Pol
- 2µl Buffer
- 15µl dH2O
- Transformation
- ligation + TOP10 --> LB(cam)
- Results of test-trafo
- Amp, Km, Cm OK --> Km with fewer colonies
- Tet --> hardly colonies
- --> comp.TOP10 obviously all right, exclusion of Tet in future work
Week 9
03.09.2012 - 09.09.2012
03.09.2012
- Results
- Transformation
- GixR in C3 --> no colonies
- CFP-T/mRFP-T in C3(31.8.) --> no colonies
- CFP-T/mRFP-T in T3(31.8.) --> many but small colonies
- CFP-T/mRFP-T in T3(29.8.) --> less colonies
- PCR
- of colonies (CFP-T, mRFP-T)
- parameters: 98°C 5', >>98°C 1', 72°C 1'<<x30, 72°C 5', 4°C
- Agarose gel
- 6x colony pT3-CFP-T from 29./31.8.
- 6x colony pT3-mRFP-T from 29./31.8.
- pC3-CFP (14)
- pC3.mRFP (24)
- result watch picture
Week 10
10.09.2012 - 16.09.2012
11.09.2012
- microscopy-screening
- GroES-GFP ⇒; no fluorescence
- GroES-CFP ⇒; no fluorescence
- AmiC-mRFP ⇒; no fluorescence
- AmiC-GFP ⇒; no fluorescence
- colony-PCR
- GixR
- RBS-Gin
- Terminator
- 3A-Assembly (digestion, ligation, transformation)
- GroES (E/S) + Terminator (X/P) + pSB1K3 (P/E)
- AmiC (E/S) + Terminator (X/P) + pSB1K3 (P/E)
- plasmid-prep and control-digestion (E/P)
- GroES-GFP
- GroES-CFP
- AmiC-mRFP
- AmiC-GFP
- GroES-Terminator
- Enhancer
- GFP-Terminator
- Terminator
