Team:Heidelberg/Templates/DelH week9

From 2013.igem.org

Contents

24-06 - 30-06-13

Characterization of DelH plasmid 19-06

SDS Page

  • Using NuPAGE Novex 10% Bis-Tris precast gel from Invitrogen with MOPS running buffer
3NuPAGE Novex 3-8% Tris-Acetate precast gel would have been more suitable considering expected 600 kDa DelH, but needed Tris-Acetate SDS buffer was not available.
  • Resuspended pellets in 100 µl SDS buffer (from Linda)
  • Boiled for 10 min @98°C
  • Ran for 90 min, 180 V
  • Stained for 40 min in Coomassie Buffer (50 ml acetic acid 100% + 225 ml ddH2O, 1.5 g Coomassie in 225 ml methanol, mix both)
  • Washed in ddH2O multiple times

Result

Fig.9.1 SDS PAGE
L1: Ladder L2: 6 µl of Konrad’s E. coli L3: 6 µl DelH colony 4 L4: 6 µl DelH colony 6 L5: 9 µl of Konrad’s E. coli L6: 9 µl DelH colony 4 L7: 9 µl DelH colony 6 L8: 15 µl of Konrad’s E. coli L9: 15 µl DelH colony 4 L10: 15 µl DelH colony 6 L11: empty L12: Ladder

None clear band at ~600 kDa visible. Maybe in highest amounts, but not reliable.

=> Probably no DelH expression in analyzed colonies.


Mini Prep

  • Picked colonies from LB Amp plates into 5 ml LB Amp (colonies 4, 6 and 9)
  • Incubation ON at 37°C
  • 5 ml ON cultures were mini prepared and resuspended in 50 µl

Test Restriction Digest

  • 5 µl of the mini prep were test digested
Reagent Volume [µl]
Miniprep DNA 5
CutSmart buffer 2
EcoRI-HF, PacI, KpnI-HF 0.5 each
ddH2O 10.75
  • Incubation at 37°C for 1 h

Result

Expected band pattern: 5 kp, 7.3 kp, 13 kp

Fig.9.2 Gel of amplified fragments (loaded 20 µL)
l1: 2 log ladder, l2:test digested DelH fragment
l2: no visible band

No clear bands visible.

=> DNA yield from mini prep was probably too low.

DNA Concentration

Colony DNA concentration [ng/µl]
1 124.5
4 3.9
6 1.6
9 10.2

DNA measurement confirmed suspicion from test digest.

=>Thus, mini prep and test digest have to be repeated


Repetion of Mini Prep

  • Picked colonies from LB Amp plates into 5 ml LB Amp (colonies 4, 6 and 9) or from remaining ON culture (colony 1)
  • Incubation ON at 37°C
  • Colony 1 did not grow (also not on LB Amp plate), obviously lost plasmid
  • Mini preped remaining 3, eluted in 35 µl ddH2O

DNA Concentration

Colony DNA concentration [ng/µl]
4 38.5
6 42.0
9 49.0

Test Restriction Digest

  • Digested entire mini prep (using remaining from first mini for colony 1) for 2 h at37°C
Reagent Volume [µl]
Mini prep 42
CutSmart 5
EcoRI-HF, PacI, KpnI-HF 1 each

Result

Expected bands: BB (7.3 Kb), DelH F2 (8 Kb), F1a + F1b (10 Kb)

Fig.9.3 miniprep resitriction digested (loaded 20 µL)
l1: 2 log ladder, l2: DelH-F1a
l2: DelH-F1a shows specific band = cut out

Unexpected bands at 3 Kb, but not desired ones.

=> Colonies do not contain desired plasmid and entire ligation has to be repeated.


Amplification of DelH F1b

PCR Conditions F1b.W9.A

Reagent DelH F1b
Template 1 µl D. acidovorans stock 29-04-13
Primer fw 10 µM 0.5 µl DelH_EcoRI_fw 100 µM
Primer rev 10 µM 0.5 µl DelH_f1_SalI_rev 100 µM
Phusion Ready Mix 25 µl
ddH2O 23 µl
Cycles Temperature [°C] Time
1 98 30 s
35 98 15 s
68 5 s
72 3:30 min
1 72 10 min
1 4 inf
  • Using hot start at 98°C

Result

Expected band: 5 Kb, Loaded 1 µl of PCR

Fig.9.4 gel of amplified DelH 1b - fragment (loaded 20 µL)
l1: 2 log ladder, l2: DelH-F1b
l2: DelH-F1b shows no band

No correct band visible.

=> Why could we not reproduce the previous amplification?


PCR Conditions F1b.W9.B

Reagent DelH F1b
Template 1 µl D. acidovorans stock 29-04-13
Primer fw 10 µM 0.5 µl DelH_EcoRI_fw 10 µM
Primer rev 10 µM 0.5 µl DelH_f1_SalI_rev 10 µM
Phusion Ready Mix 25 µl
ddH2O 23 µl
Cycles Temperature [°C] Time
1 98 30 s
30 98 15 s
68 15 s
72 3:30 min
1 72 10 min
1 4 inf
  • Using hot start at 98°C

Result

Expected band: 5 Kb, Loaded 1 µl of PCR

Fig.9.5 gel of amplified DelH-1b-fragment (loaded 20 µL)
l1: 2 log ladder, l2: DelH-F1b
l2: DelH-F1b shows unexpected band at 2.3 Kb

Again, expected band not there.

=> Adapt PCR conditions and ask for polymerase used last time, which is the one from the lab upstairs.


PCR Conditions F1b.W9.C

Reagent DelH F1b
Template 1 µl DelH F1b Fragment
Primer fw 10 µM 0.5 µl DelH_EcoRI_fw 10 µM
Primer rev 10 µM 0.5 µl DelH_f1_SalI_rev 10 µM
Phusion Flash Ready Mix 10 µl
ddH2O 7 µl
Cycles Temperature [°C] Time
1 98 10 s
30 98 1 s
68 5 s
72 120
1 72 10 min
1 4 inf
  • Using hot start at 98°C

Result

Expected band: 5 Kb, Loaded 1 µl of PCR

Fig.9.9 gel of amplified fragments (loaded 20 µL)
l1-3:F1b, l4: 2log ladder, l5-7: BB
BB was not amplified
Fig.9.8 gel of amplified fragments (loaded 20 µL)
l1-3:F1b, l4: 2log ladder, l5-7: BB
BB was not amplified
Fig.9.7 Gel of amplified DelH-fragments(F1a & F2) and Backbone (pSB6A1-AraC-lacZ) (loaded 20 µL)
l1:2log ladder, l2: F1a, l3:F2, l4: BB
l2: F1a shows specific band at 5 Kb = was cut out
Fig.9.6 Gel of amplified DelH-fragments(F1a & F2) and Backbone (pSB6A1-AraC-lacZ) (loaded 20 µL)
l1:2log ladder, l2: F1a, l3:F2, l4: BB
l2: F1a shows specific band at 5 Kb

Highly specific band at 5 Kb, as well as additional smaller ones.

=> Band was cut. Run gel with remaining sample for gel extraction.



PCR Conditions F1b.W9.D

Reagent DelH F1b
Template 1 µl D. acidovorans stock 29-04-13 1:10
Primer fw 10 µM 0.5 µl DelH_EcoRI_fw 10 µM
Primer rev 10 µM 0.5 µl DelH_f1_SalI_rev 10 µM
Q5 Ready Mix 10 µl
ddH2O 7 µl
Cycles Temperature [°C] Time
1 98 10 s
30 98 1 s
68 5 s
72 2:30 min
1 72 10 min
1 4 inf
  • Using hot start at 98°C

Result

Expected band: DelH F1b 5 Kb

Fig.9.10 gel of amplified fragments using Q5 (loaded 1 µL)
l1:2log ladder, l5: fragment 1b, l6: fragment 2, l7: BB
no product

Neither of the PCRs yield any product.

=> Therefore, Q5 is no option for us!


Amplification of DelH F2

PCR Conditions F2.W9.A

Reagent DelH 2
Template 1 µl D. acidovorans stock 29-04-13
Primer fw 10 µM 0.5 µl DelH_f2_SalI_fw 100 µM
Primer rev 10 µM 0.5 µl DelH_f2_KpnI_rev 100 µM
Phusion Ready Mix 25 µl
ddH2O 23 µl
Cycles Temperature [°C] Time
1 98 30 s
35 98 15 s
66 5 s
72 3:30 min
1 72 10 min
1 4 inf
  • Using hot start at 98°C

Result

Expected band: 8 Kb

Fig.9.11 gel of amplified fragments (loaded 1 µL)
l1: 2log ladder, l2:F1b, l3: F2, l4: BB
shows band at 3 Kb

Unexpected bands at 3 Kb in lanes of DelH 2

=> Why could we not reproduce the previous amplification?


PCR Conditions F2.W9.B

Reagent DelH 2
Template 1 µl D. acidovorans stock 29-04-13
Primer fw 10 µM 0.5 µl DelH_f2_SalI_fw 10 µM
Primer rev 10 µM 0.5 µl DelH_f2_KpnI_rev 10 µM
Phusion Ready Mix 25 µl
ddH2O 23 µl
Cycles Temperature [°C] Time
1 98 30 s
12 98 15 s
66 decrease by 0.5 15 s
72 3:30 min
18 98 15 s
66 15 s
72 3:30 min
1 72 10 min
1 4 inf
  • Using hot start at 98°C

Result

Expected band: 8 Kb

Fig.9.12 gel of amplified fragments (loaded 1 µL)
l1:2log, l2: F1b, l3: F2, l4: BB
F2 shows no specific band

Unexpected bands at 1.5 Kb, 2.2 Kb, 4 Kb and 6 Kb. Desired 8 Kb band is present but hardly visible.

=> Adapt PCR conditions and ask for polymerase used last time, which is the one from the lab upstairs.


PCR Conditions F2.W9.C

Reagent DelH 2
Template 1 µl D. acidovorans stock 29-04-13 1:10
Primer fw 10 µM 0.5 µl DelH_f2_SalI_fw 10 µM
Primer rev 10 µM 0.5 µl DelH_f2_KpnI_rev 10 µM
Phusion Flash Ready Mix 10 µl
ddH2O 7 µl
Cycles Temperature [°C] Time
1 98 10 s
12 98 1 s
66 decrease by 0.5 5 s
72 2:30 min
18 98 1 s
66 5 s
72 2:30 min
1 72 10 min
1 4 inf
  • Using hot start at 98°C

Result

Expected band: 8 Kb

Fig.9.9 Gel of amplified DelH-fragments(F1a & F2) and Backbone (pSB6A1-AraC-lacZ) (loaded 19 µL)
l1:2log ladder, l2: F1a, l3:F2
l3: F2 shows specific band at 8 Kb and other ones
Fig.9.8 Gel of amplified DelH-fragments(F1a & F2) and Backbone (pSB6A1-AraC-lacZ) (loaded 19 µL)
l1:2log ladder, l2: F1a, l3:F2
l3: F2 shows specific band at 8 Kb and other ones
Fig.9.7 Gel of amplified DelH-fragments(F1a & F2) and Backbone (pSB6A1-AraC-lacZ) (loaded 1 µL)
l1:2log ladder, l2: F1a, l3:F2, l4: BB
l2: F1a shows specific band at 8 Kb = was cut out
Fig.9.6 Gel of amplified DelH-fragments(F1a & F2) and Backbone (pSB6A1-AraC-lacZ) (loaded 1 µL)
l1:2log ladder, l2: F1a, l3:F2, l4: BB
l3: F2 shows specific band at 8 Kb and other ones

F2 shows specific band at 8 Kb and additional ones.

=> Band was cut. Run gel with remaining sample for gel extraction.


PCR Conditions F2.W9.D

Reagent DelH 2
Template 1 µl D. acidovorans stock 29-04-13 1:10
Primer fw 10 µM 0.5 µl DelH_f2_SalI_fw 10 µM
Primer rev 10 µM 0.5 µl DelH_f2_KpnI_rev 10 µM
Q5 Ready Mix 10 µl
ddH2O 7 µl
Cycles Temperature [°C] Time
1 98 10 s
12 98 1 s
66 decrease by 0.5 5 s
72 2:30 min
18 98 1 s
66 5 s
72 2:30 min
1 72 10 min
1 4 inf
  • Using hot start at 98°C

Result

Expected band: 8 Kb, Loaded 1 µl of PCR

Fig.9.10 gel of amplified fragments using Q5 (loaded 1 µL)
l1:2log ladder, l5: fragment 1b, l6: fragment 2, l7: BB
no product

Neither of the PCRs yield any product.

=> Therefore, Q5 is no option for us!


Amplification of Backbone pSB6A1-AraC-lacZ

PCR Conditions BB.W9.A

Reagent Backbone
Template 1 µl Backbone (pSB6A1-AraC-lacZ c = 24 ng/µl)
Primer fw 10 µM 0.5 µl AraCbb_KpnI_fw 100 µM
Primer rev 10 µM 0.5 µl AraCbbPacI_rev2 100 µM
Phusion Ready Mix 25 µl
ddH2O 23 µl
Cycles Temperature [°C] Time
1 98 30 s
35 98 15 s
66 5 s
72 3:30 min
1 72 10 min
1 4 inf
  • Using hot start at 98°C

Result

Expected band: 7.3 Kb, Loaded 1 µl of PCR

Fig.9.11 Gel of amplified fragments of DelH and BB(loaded 1 µL)
l1:2log ladder, l5: F1b, l6: F2, l7: BB
no product

No band visible, amplification failed.

=> Why could we not reproduce the previous amplification?


PCR Conditions BB.W9.B

Reagent BB
Template 1 µl Backbone (pSB6A1-AraC-lacZ c = 24 ng/µl)
Primer fw 10 µM 0.5 µl AraCbb_KpnI_fw 10 µM
Primer rev 10 µM 0.5 µl AraCbbPacI_rev2 10 µM
Phusion Ready Mix 25 µl
ddH2O 23 µl
Cycles Temperature [°C] Time
1 98 30 s
12 98 15 s
66 decrease by 0.5 15 s
72 3:30 min
18 98 15 s
66 15 s
72 3:30 min
1 72 10 min
1 4 inf
  • Using hot start at 98°C

Result

Expected band: 7.3 Kb, Loaded 1 µl of PCR

Fig. 9.12 Gel of amplified DelH-fragments and BB l1:2log, l2: F1b,l3:F2, l4:BB

Unexpected band at 2.2 Kb

=> Adapt PCR conditions and ask for polymerase used last time, which is the one from the lab upstairs.


PCR Conditions BB.W9.C

Reagent DelH 2
Template 1 µl pSB6A1-AraC-LacZ digested and purified 1:10
Primer fw 10 µM 0.5 µl AraCbb_KpnI_fw 10 µM
Primer rev 10 µM 0.5 µl AraCbbPacI_rev2 10 µM
Phusion Flash Ready Mix 10 µl
ddH2O 7 µl
Cycles Temperature [°C] Time
1 98 10 s
12 98 1 s
66 decrease by 0.5 5 s
72 2:30 min
18 98 1 s
66 5 s
72 2:30 min
1 72 10 min
1 4 inf
  • Using hot start at 98°C

Result

Expected band: 7.3 Kb, Loaded 1 µl of PCR

Fig.9.6 Gel of amplified DelH-fragments(F1a & F2) and Backbone (pSB6A1-AraC-lacZ) (loaded 20 µL)
l1:2log ladder, l2: F1a, l3:F2, l4: BB
l4: BB shows no expected band

Gel does not show expected fragment.

=> Make sure right template was used. Is the restricted and purified fragment suitable?


PCR Conditions BB.W9.D

Reagent DelH 2
Template 1 µl pSB6A1-AraC-LacZ digested and purified 1:10
Primer fw 10 µM 0.5 µl AraCbb_KpnI_fw 10 µM
Primer rev 10 µM 0.5 µl AraCbbPacI_rev2 10 µM
Q5 Ready Mix 10 µl
ddH2O 7 µl
Cycles Temperature [°C] Time
1 98 10 s
12 98 1 s
66 decrease by 0.5 5 s
72 2:30 min
18 98 1 s
66 5 s
72 2:30 min
1 72 10 min
1 4 inf
  • Using hot start at 98°C

Result

Expected band: 7.3 Kb, Loaded 1 µl of PCR

Fig.9.10 gel of amplified fragments using Q5 (loaded 1 µL)
l1:2log ladder, l5: fragment 1b, l6: fragment 2, l7: BB
no product

Neither of the PCRs yield any product.

=> Therefore, Q5 is no option for us!