Team:Heidelberg/Templates/DelH week14

From 2013.igem.org

Revision as of 11:19, 25 October 2013 by Anja R (Talk | contribs)

Contents

29-07 - 04-08-13

Generation of DelH Plasmid 28-07

Purification of Gibson Mix

10 µl of the Gibson mix were isopropanol purified as described in the protocol.
5 µl of the Gibson reaction were mixed with 10 µl ddH2O.

Electroporation

  • Electroporation into freshly prepared electrocompetent cell following the protocol.
E1 E2 E3
5 µl of Gibson + 10 µl H2O 10 µl of isoprop purified Gibson assembly no DNA
  • Grow 0.5 h with 400 µl SOC at 37°C
  • Add 9 ml LB Amp and incubate 3 h at 37°C
  • Incubate ON at 37°C on LB Amp plate as well as LB (no Amp) for control purposes

Result

There were few colonies on the plates. Red colonies were picked and screenied by colony-PCR, but none of them was positive. The Gibson assembly has to be repeated.

Generation of DelH Plasmid 01-08

Gibson Assembly

Fragment Concentraion Amount used [µl] (2 vials) Amount used [µl]
G0 0,68 ng/µl 9 6
BB (8.8) 22 ng/µl 1 4
Gibson Master Mix 2x 10 10
  • Incubated 1 h at 50 °C in thermocycler

Electroporation

Electroporation # Mix # Amount of Mix [µl] ddH2O [µl] Isopropanol Amount electroporated [µl]
1 1 10 20 - 1
2 1 10 20 - 14
3 1 30 - yes 20
4 2 5 10 - 1
5 2 5 10 - 29
6 2 15 - yes 20
7 - - 10 - 10
  • Plated on one LB agar plate each
  • Stored ON at 37°C


Colony-PCR Conditions CP.W14.A

Reagent Electroporation 1 Electroporation 2 Electroporation 3 Electroporation 4 Electroporation 5 Electroporation 6
Expected length [bp] 663 663 663 663 663 663
Named 1 2 3 4 5 6
Template 5 colonies of E1 5 colonies of E2 5 colonies of E3 5 colonies of E4 5 colonies of E5 5 colonies of E6
Primer fw 10 µM 1 µl VF2 1 µl VF2 1 µl VF2 1 µl VF2 1 µl VF2 1 µl VF2
Primer rev 10 µM 1 µl Screen_delH_rev 1 µl Screen_delH_rev 1 µl Screen_delH_rev 1 µl Screen_delH_rev 1 µl Screen_delH_rev 1 µl Screen_delH_rev
Dream-Taq Polymerase (2x) 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl
ddH2O 8 µl 8 µl 8 µl 8 µl
Cycles Temperature [°C] Time [s]
1 95 120
12 95 60
68 (touchdown -0.5°C) 30
72 45
18 95 60
65 (touchdown -0.5°C) 30
72 45
1 12 inf

Result

Expected band: 663 bp

Fig.14.1 Gel of picked colonies with insert DelH-BB (loaded 20 µL of PCR)
l1: 1, l2: 2,l3: 3, l4: 2log,l5: 4, l6: 5,l7: 6
no bands = no colony positive

Gel does not show any band.

=> None of the analyzed colonies is positive.


Colony-PCR Conditions CP.W14.B

Picked 20 colonies per plate and 5 colonies per PCR tube.

Reagent Electr. 1 Electr. 1 Electr. 1 Electr. 2 Electr. 2 Electr. 2 Electr. 2
Expected length [bp] 663 663 663 663 663 663 663
Named 1a 1b 1c 2a 2b 2c 2d
Template 5 colonies of E1 5 colonies of E1 5 colonies of E1 5 colonies of E2 5 colonies of E2 5 colonies of E2 5 colonies of E2
Primer fw 10 µM 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2
Primer rev 10 µM 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev
Dream-Taq Polymerase (2x) 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl
ddH2O 6 µl 6 µl 6µl 6 µl 6 µl 6 µl 6 µl
Reagent Electr. 3 Electr. 3 Electr. 3 Electr. 3 Electr. 4 Electr. 4 Electr. 4 Electr. 4
Expected length [bp] 663 663 663 663 663 663 663 663
Named 3a 3b 3c 3d 4a 4b 4c 4d
Template 5 colonies of E3 5 colonies of E3 5 colonies of E3 5 colonies of E3 5 colonies of E4 5 colonies of E4 5 colonies of E4 5 colonies of E4
Primer fw 10 µM 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2
Primer rev 10 µM 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev
Dream-Taq Polymerase (2x) 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl
ddH2O 6 µl 6 µl 6 µl 6 µl 6 µl 6µl 6 µl 6 µl
Reagent Electr. 5 Electr. 5 Electr. 5 Electr. 5 Electr. 6 Electr. 6 Electr. 6 Electr. 6
Expected length [bp] 663 663 663 663 663 663 663 663
Named 5a 5b 5c 5d 6a 6b 6c 6d
Template 5 colonies of E5 5 colonies of E5 5 colonies of E5 5 colonies of E5 5 colonies of E6 5 colonies of E6 5 colonies of E6 5 colonies of E6
Primer fw 10 µM 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2
Primer rev 10 µM 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev
Dream-Taq Polymerase (2x) 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl
ddH2O 6 µl 6 µl 6 µl 6 µl 6 µl 6 µl 6 µl 6 µl
Cycles Temperature DelH-G0 [°C] Time [s]
1 95 120
12 95 60
68 (touchdown -0.5°C) 30
72 45
18 95 60
65 (touchdown -0.5°C) 30
72 45
1 12 inf


Result

Expected band: 663 bp

Fig.14.3 Gel of picked colonies with insert DelH-BB (loaded 20 µL of PCR)
l1: 2log, l2-5: 20 picked colonies from plate 4: every PCR tube has 5 colonies, l6-9:20 picked colonies from plate 5every PCR tube has 5 colonies,l10-14:20 picked colonies from plate 6 every PCR tube has 5 colonies
bands on lanes 4a, 4b, 6a, 6b, 6d at the expected 663 bp, so the next step is making PCRs of 1 colony per PCR of these probes
Fig.14.2 Gel of picked colonies with insert DelH-BB (loaded 20 µL of PCR)
l1-3:3 picked colonies from plate 1, every PCR tube has 5 colonies, l4-7:4 picked colonies from plate 2, every PCR tube has 5 colonies,l8: 2log,l9-13:4 picked colonies from plate 3 every PCR tube has 5 colonies
bands on lanes 3b, 3c, 3d, 4a, 4b, 6a, 6b, 6d at the expected 663 bp, so the next step is making PCRs of 1 colony per PCR of these probes

Gel shows bands for b, 3c, 3d, 4a, 4b, 6a, 6b, 6d at the expected 663 bp.

=> Make single colony-PCRs.


Colony-PCR Conditions CP.W14.C

1 µl of colony ON culture picked

Reagent 3c 3c 3c 3c 3c 4a 4a 4a 4a 4a 6b 6b 6b 6b 6b
Expected length [bp] 663 663 663 663 663 663 663 663 663 663 663 663 663 663 663
Named 3c 1 3c 2 3c 3 3c 4 3c 5 4a 1 4a 2 4a 3 4a 4 4a 5 6b 1 6b 2 6b 3 6b 4 6b 5
Template (1 µl) 1 colonies of E3c 1 colonies of E3c 1 colonies of E3c 1 colonies of E3c 1 colonies of E3c 1 colonies of E4a 1 colonies of E4a 1 colonies of E4a 1 colonies of E4a 1 colonies of E4a 1 colonies of E6b 1 colonies of E6b 1 colonies of E6b 1 colonies of E6b 1 colonies of E6b
Primer fw 10 µM 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2
Primer rev 10 µM 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev
Dream-Taq Polymerase (2x) 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl
ddH2O 5 µl 5 µl 5µl 5 µl 5 µl 5 µl 5 µl 5 µl 5 µl 5 µl 5 µl 5 µl 5µl 5 µl 5 µl
Cycles Temperature DelH-G0 [°C] Time [s]
1 95 120
12 95 60
68 (touchdown -0.5°C) 30
72 45
18 95 60
65 (touchdown -0.5°C) 30
72 45
1 12 inf

Result

Expected band: 663 bp

Fig.14.5 Gel of picked colonies with insert DelH-BB (loaded 20 µL of PCR)
'l1: 2log, l2-6: 5 seperate colonies which were together in 6b PCR earlier,
no bands = no colony positive
Fig.14.4 Gel of picked colonies with insert DelH-BB (loaded 20 µL of PCR)
'l1: 2log, l2-6: 5 seperate colonies which were together in 3a PCR earlier, l7-11: 5 seperate colonies which were together in 4b PCR earlier,
no bands = no colony positive


=> No band is visible. There should be a band at at least one of each sample (for example 6B), because in the previous PCR we observed a band.


Because of the explained reason above we incubated 40 colonies seperately which might be positive ON at 37°C in 200 µl LB Amp. 200 µl of the ON cultire was mixed with 1.8 ml LB Amp for five colonies of each probe: 3b, 3c, 3d, 4a, 4b, 6a, 6b, 6d.

Retrieved from "http://2013.igem.org/Team:Heidelberg/Templates/DelH_week14"