Team:Freiburg/Notebook/lab gfp reporter

From 2013.igem.org

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<h2> 10.05.13 </h2>
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<h3>Fusion PCR</h3>
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<h3> Fusion PCR </h3>
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<h4>1. CMV+eGFP</h4>
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<h4> 1. CMV+eGFP </h4>
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Revision as of 17:50, 24 September 2013


GFP-reporter-plasmid Notebook

May

03. May 2013

Testdigest of pSB1C3 backbone

As a source plasmid to get the pSB1C3 backbone we used a biobrick called BBa_K515107

Approach of test digest:

µl
2 BBa_K515107
2 NEB Buffer 4 (10X)
0.5 EcoR1-HF
0.5 Pst1-HF
15 H2O

PCR 1

µl type
25 Q5-HF Mastermix
20ng DNA
2.5 Primer1
2,5 Primer2
Add to 50 H2O
  1. CMV-promotor
    • oIG6000
    • oIG6001
    • Temp.: eGFP-Plasmid
  2. GFP
    • oIG6002
    • oIG 6003
    • Temp.: eGFP-Plasmid
  3. Terminator
    • oIG6004
    • oIG6005
    • Temp.: eGFP-Plasmid
  4. CFP
    • oIG6002
    • oIG6012
    • Temp.: CFP M4 Plasmid

Gel of PCR 1

expected length of the fragments: 1. CMV: 685bp 2. eGFP: 824bp 3.terminator: 350bp 4.CFP: 820bp bands were not expactly running as expected, but perhaps due to the marker. the marker bands are not separated clearly. So the bands were cut out and gel ex was performed. Nanodropping after the Gel ex: 1. CMV: 156ng/µl 2. eGFP: 143ng/µl 3. terminator: 163ng/µl 4. CFP: 85ng/µl

07. May 3013

Fusion PCR 7.5.13

µl type
10 5x Q5 Buffer 
4 DNTPs
0.5 Q5
1 CMV
1 eGFP
2.5 oIG600
2,5 oIG6003
Add to 50 H2O
µl type
10 5x Q5 Buffer 
4 DNTPs
0.5 Q5
1 terminator
1.5 CFP
2.5 oIG602
2,5 oIG6005
Add to 50 H2O

additionally a no template control was performed. the PCRs were run with Manus PCR programm. 1.30min elongation time.

preperation digest of pSB1C3

As a source plasmid to get the pSB1C3 backbone we used a biobrick called BBa_K515107

Approach of test digest:

µl
5 BBa_K515107
5 NEB Buffer 4 (10X)
1 Xba-HF
1 Pst1-HF
38 H2O

gelbild of fusion PCR and prep digest

08. May 3013

PCR: second try of the first one

µl type
10 Q5-Buffer
4 DNTPs
2 DNA (1:10 diluted)
0,5 Q5
2.5 Primer fw
2,5 Primer rv
Add to 50 H2O

the same results as in the first try were obtained and the bands were cut out and put in the freezer but a gel ex was never performed.

digest of pSB1C3 (Bba_K32305 and Bba_K32380)

µl type
2 DNA
5 NEB-Buffer 4
1 Xba1
1 Pst1
Add to 50µl H2O
  • Temp.: 37°C
  • Incubation time: 2h

length of backbone: 2070bp. insert that is cut out: 1274bp (...80). Bands on gel as expected.

10.05.13

Fusion PCR

1. CMV+eGFP

µl type
10 Q5-HF Reaction Buffer
1 CMV
1 eGFP
2,5 oIG6000
2,5 oIG6003
2.5 dNTPs
1 DMSO
Add to 50 H2O

2. CFP+SV40 terminator

µl type
10 Q5-HF Reaction Buffer
1 ecfp
1 termin
2,5 oIG6002
2,5 oIG6005
2.5 dNTPs
1 DMSO
Add to 50 H2O

3. eGFP+ SV40 terminator

µl type
10 Q5-HF Reaction Buffer
1 termin
1 eGFP
2,5 oIG6002
2,5 oIG6005
2.5 dNTPs
1 DMSO
Add to 50 H2O
  • Annealing: 60°C
  • Extension:40s

June

5.6.13

PCR of construct 405 (Anne & Patrick) for CMV promotor

µl type
10 Q5-HF Reaction Buffer
1 Template: 405
2,5 oIG6000
2,5 oIG6001
4 dNTPs
0,5 Q5-HF Polymerase
Add to 50 H2O
  • Annealing: 60°C
  • Extension:

PCR of construct 405 (Anne & Patrick) for GFP

µl type
10 Q5-HF Reaction Buffer
1 Template: 405
2,5 oIG6002
2,5 oIG6003
4 dNTPs
0.5 Q5-HF Polymerase
Add to 50 H2O
  • Annealing: 60°C
  • Extension:

6.6.13

Gelelectrophoresis of PCR products (5.6.13)

Gelextraction

name ng/µl
GFP (405) 88
CMV (405) 98
Terminator 36
pSB1C3 05 (old) 26,7

Gibson

µl type
0,92 pSB1C3
0,9 GFP
0,66 CMV
0,47 Terminator
2,06 H2O
  • 1 h,50 °C
  • 3 min. RT
  • 3 min, 4 °C
  • 4 µl Transformation

7.6.13

Testdigest:

µl type
1 and 2 DNA
1 NEB-Buffer 4
0.5 EcoRI
0.5 BamHI
Add to 10 µl H2O
  • Temp.: 37°C
  • Incubation time: 1,5h

PCR of eCFP

µl type
10 Q5-HF Reaction Buffer
1 Template: Pal113 (eCFP)
2,5 oIG6025
2,5 oIG6002
4 dNTPs
0,5 Q5-HF Polymerase
Add to 50 H2O
  • Annealing: 60°C
  • Extension:

PCR of bgh Terminator

µl type
10 Q5-HF Reaction Buffer
1 Template: pMH (bgh terminator)
2,5 oIG6022
2,5 oIG6023
4 dNTPs
0,5 Q5-HF Polymerase
Add to 50 H2O
  • Annealing: 60°C
  • Extension:20sec

10.6.13

Repetition of PCR of eCFP

  • temperature gradient: 48°C - 60°C
µl type
10 Q5-HF Reaction Buffer
1 Template: Pal113 (eCFP)
2,5 oIG6002
2,5 oIG6003
4 dNTPs
1,5 DMSO
0,5 Q5-HF Polymerase
Add to 50 H2O
Tube temperature [°C]
1 48
2 50
3 51,4
4 52,9
5 56,4
6 58,7
7 60

Sequencing of Minipreps of pIG6000 (1 & 2) (8.6.13)

Gibson: CMV, GFP (405), bgh (105) and pSB1C3

µl type
0,92 pSB1C3
0,9 GFP
0,66 CMV
0,56 bgh Terminator
add to 5 µl H2O
  • 1 h,50°C
  • 3 min. RT
  • 3 min,°C
  • 4 µl Transformation

Gibson: CMV (405), eCFP (Pal113), bgh (105) and pSB1C3

µl type
0,92 pSB1C3
1,36 eCFP
0,66 CMV
0,56 bgh Terminator
add to 5 µl H2O
  • 1 h,50°C
  • 3 min. RT
  • 3 min, 4°C
  • 4 µl Transformation

11.6.13

Gibson has worked: 6 colonies for minipreps

Testdigest: Gibson (6.6.13)

µl type
1 DNA
1 NEB-Buffer 4
0.5 NdeI
0.5 XhoI
Add to 10 µl H2O
  • Temp.: 37°C
  • Incubation time: 1,5h

the testdigest showd as expected 3 bands (896bp, 389bp ans 2517bp) Bands of clone 8 seemed to be right and we sent it for sequenzing (primer: 17, 18)

12.6.13

Sequnecing of clone 8 containing CFP were unclear. to test if the two plasmid are functional they were transfected into CHO cells. therefore 750ng DNA of each mini-prep were transfected per well in a 48 well plate.

13.6.13

In parallel to the transfection test a testdigest of piG6000 and piG6001 was performed.

Testdigest: piG6000 and piG6001

µl type
1 DNA
2 NEB-Buffer 4
0.5 NdeI
0.5 XhoI
Add to 10 µl H2O
  • Temp.: 37°C
  • Incubation time: 1,5h

clone 3 and 4 of piG6000 run as expected and they also show strong fluorescence in the CHO cells. Clone c,d,e and f of piG6001 run higher as expected but they do not show visible flourescence (this might be due to transfecting a mini-prep instead of a midi-prep) Clone 3 of piG6000 and clone e of piG6001 were sequenced

18.13

sequencing results of both constructs are positive.

18.13

midi-prep of pIG600 and retrafo of pIG6001

20.13

CMV min

Idea: making pIG6000 able to be activated by Cas9-VP16. therefore the constitutively active CMV promoter is exchanged by a CMV min. oligos will be designed to target PsB1C3 ao that VP16 can activate the minimal promoter and the cells start to show green fluorescence.

digest of pIG6000

µl type
10 DNA
5 NEB-Buffer cut smart
2,5 NheI HF
12,5 SacII
Add to 50µl H2O
  • Temp.: 37°C
  • Incubation time: 1,5h

digest of plasmid of Patrick and Anne (600)

µl type
10 DNA
5 NEB-Buffer cut smart
2,5 NheI HF
12,5 SacII
Add to 50µl H2O
  • Temp.: 37°C
  • Incubation time: 1,5h

Bands were as expected:
pIG6000: 631bp and 3154bp. here the higher band was cut out as it is the vector
pIGPanne: 109bo and 2683bp. here the lower, smaler band was cut out as it is the CMVmin prompter. Gel ex was performed

Ligation of pIG6002

µl type
7 vector
5 ,5 Insert
2 T4 ligase Buffer
1 T4 ligase
Add to 50µl H2O
  • Temp.: 16°C
  • Incubation time: 1h

Then E.Coli were transformed with 4µl of the ligation.

22.6.13

Mini-preps of pIG6002 were performed

23.13

test digest of pIG6002

µl type
4 DNA
52 NEB-Buffer cut smart
0,5 NheI HF
0,5 SacII
Add to 20µl H2O
  • Temp.: 37°C
  • Incubation time: 1,5h

all 6 digested mini showed the same positive result! therefore the CMVmin project is accomplished

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