Team:Heidelberg/Templates/DelH week14

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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
Expected length [bp] 663 663 663 663 663
Named 3c 1 3c 2 3c 3 3c 4 3c 5
Template (1 µl) 1 colonies of E3c 1 colonies of E3c 1 colonies of E3c 1 colonies of E3c 1 colonies of E3c
Primer fw 10 µM 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
Dream-Taq Polymerase (2x) 10 µl 10 µl 10 µl 10 µl 10 µl
ddH2O 5 µl 5 µl 5µl 5 µl 5 µl
Reagent 4a 4a 4a 4a 4a
Expected length [bp] 663 663 663 663 663
Named 4a 1 4a 2 4a 3 4a 4 4a 5
Template (1 µl) 1 colonies of E4a 1 colonies of E4a 1 colonies of E4a 1 colonies of E4a 1 colonies of E4a
Primer fw 10 µM 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
Dream-Taq Polymerase (2x) 10 µl 10 µl 10 µl 10 µl 10 µl
ddH2O 5 µl 5 µl 5 µl 5 µl 5 µl
{| class="wikitable" style="float:left; margin-right:1em"

|- ! Reagent !! 6b !! 6b !! 6b !! 6b !! 6b |- | Expected length [bp] || 663 || 663 || 663 || 663 || 663 |- | Named ||6b 1 || 6b 2 || 6b 3 || 6b 4 || 6b 5 |- | Template (1 µl) || 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 |- | 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 |- | Dream-Taq Polymerase (2x) || 10 µl || 10 µl || 10 µl || 10 µl || 10 µl |- | ddH2O || 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.

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