Team:Heidelberg/Templates/DelH week6

From 2013.igem.org

Contents

03-06 - 09-06-13

Generation of DelH plasmid pHM01

Elongation-PCR Conditions BB.W6.A

The restriction sites were added to pSB6A1-AraC-lacZ via PCR.

Reagent pSB6A1-AraC-lacZ
Expected length [Kb] 7.4
Template 1 µl of 1:10 pSB6A1-AraC-lacZ miniprep
Primer fw 10 µM DN05
Primer rev 10 µM DN06
Phusion Master Mix (2x) 25 µl
DMSO -
ddH2O 23 µl
Cycles Temperature [°C] Time [s]
1 98 10
14 98 1
66↓ 5
72 2:00 min
16 98 1
66 5 s
72 2:00 min
1 72 2:30 min
1 4 inf

Result

Expected band: 7.4 Kb

Fig.6.1 PCR of pSB6A1-AraC-lacZ & digest of pSB6A1-AraC-lacZ (loaded 5 & 20 µL of PCR)
l1: 2log ladder, l2-3: PCR of BB, l4-5: digested BB of EcoRI & PstI
PCR and digest sho the expected band at 7.4 Kb but also unspecific side bands


Gel shows expected band.

=> Band was cut and gel extracted.


Test Restriction Digest

Isolated fragment was test digested to confirm identity.

Sample Concentration [ng/µl] Volume for digest [µl] Enzyme [µl] Buffer [µl] ddH2O [µl] Final volume [µl]
pSB6A1-AraC-lacZ 99 5 1.5 EcoRI-HF & 1.5 PstI 5 NEB2 & 5 BSA 32 50
  • The test digest was incubated 1 h at 37°C.

Result

Expected band: ~4 Kb & 3.4 Kb

Fig.6.1 Digest of pSB6A1-AraC-lacZ (loaded 5 & 20 µL of PCR)
l1: 2log ladder, l2-3: PCR of BB, l4-5: digested BB of EcoRI & PstI
PCR and digest sho the expected band at 7.4 Kb but also unspecific side bands

Gel shows expected band.

=> Backbone is fine.


Restriction Digest of pSB6A1-AraC-lacZ for Ligation

Sample Concentration [ng/µl] Volume for digest [µl] Enzyme [µl] Buffer [µl] ddH2O [µl] Final volume [µl]
pSB6A1-AraC-lacZ 99 12.5 1.5 KpnI-HF & 1.5 PacI 5 NEB4 & 5 BSA 24.5 50

Restriction Digest was purified with Qiagen nucleotide removal kit.

Result

c=24 ng/µl

Restriction Digest of DelH F1a for Ligation

Sample Concentration [ng/µl] Volume for digest [µl] Enzyme [µl] Buffer [µl] ddH2O [µl] Final volume [µl]
DelH F1a  ? 25 1.5 EcoRI-HF & 1.5 PacI 5 NEB4 & 5 BSA 12 50

Restriction Digest was purified with Qiagen nucleotide removal kit.

Result

c=9 ng/µl

Restriction Digest of DelH F1b for Ligation

Sample Volume for digest [µl] Enzyme [µl] Buffer [µl] ddH2O [µl] Final volume [µl]
DelH F1b 25 1.5 EcoRI-HF & 1.5 SalI-HF 5 NEB4 & 5 BSA 12 50

Restriction Digest was purified with Qiagen nucleotide removal kit.

Result

c=19 ng/µl

Restriction Digest of DelH F2 for Ligation

Sample Volume for digest [µl] Enzyme [µl] Buffer [µl] ddH2O [µl] Final volume [µl]
DelH F2 25 1.5 KpnI-HF & 1.5 SalI-HF 5 NEB4 & 5 BSA 12 50

Restriction Digest was purified with Qiagen nucleotide removal kit.

Result

c=9 ng/µl

Ligation of pHM01:DelH-pSB6A1-AraC-lacZ

Ligations was performed of all 4 fragments (digested and purified) for 1 h at RT in 2 different mixes.

Sample Concentration [ng/µl] Volume for Ligation [µl]
pSB6A1-AraC-lacZ 24 2
DelH F1a 9 6
DelH F1b 19 3
DelH F2 9 6
T4 ligase - 1
Buffer - 2
ddH2O - -
Final volume - 20
  • The ligase was inactivated by heat shock for 5 min at 70°C.
  • The reaction mix was purified with Qiagen nucleotide removal.

Result

Sample Concentration [ng/µl]
1 20
2 30


Electroporation

Two electroporations were performed.

  • 20 ng
  • 30 ng
  • After an incubation time of 1 h in SOC-medium, cells were centrifuged and 10 µl plated on one LB Amp plate (with Arabinos and X-Gal) and 100 µl on another plate prepared with the same conditions.
  • Plates were incubated ON at 37°C.

Result

White colonies grew on the plates.

Colony PCR

6 colonies of plate E1 (colonies 1-6) and 6 colonies of plate E2 (7-12) as well as ligation mixes 1 & 2 were checked by PCR.

Reagent DelH colonies
Expected length [bp] 663
Primer fw 10 µM 2 µl VF2
Primer rev 10 µM 2 µl DN07
Taq Polymerase (2x) 10 µl
ddH2O 6 µl
Cycles Temperature [°C] Time
1 95 120
12 95 60
66 (touchdown -0.5°C) 30
72 45
18 95 60
62 (touchdown -0.5°C) 30
72 45
1 12 inf

Result

Expected length: 663 bp.

Fig.6.2 Colony PCR of DelH-construct (loaded 5 µL)
l1:2log ladder, l2-10:Colony PCR of colonies 1-9, l10:2 log ladder, l11-15:Colony PCR of colonies 1-9
l2-3: shows expected band at 663 bp

None of the colonies shows a band. Ligation mixes 1 and 2 show the expected band at ~600 bp.

=> The ligation worked, but none of the correct plasmids were present in the analyzed colinies.


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