Team:Heidelberg/Templates/DelH week4

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Contents

20-05 - 26-05-13

DelH Fragment F1a

  • The new primer arrived.

PCR Conditions F1a.W4.A

Reagent DelH F1a DelH F1a
Expected length [Kb] 5 5
Template 1 µl D. acidovorans 1 µl D. acidovorans
Primer fw 10 µM DN01 DN01
Primer rev 10 µM DN08 DN08
Phusion Master Mix (2x) 25 µl 25 µl
DMSO 2.5 µl -
ddH2O 20,5 23 µl
Cycles Temperature [°C] Time [s]
1 98 10
16 98 1 s
62 5 s
72 2:00 min
1 72 2:30 min
1 4 inf

Results

Expected band: 5 Kb

Fig.4.1 PCR ofDelH-fragments and pSB1C3(loaded 20 µL)
l1:2log ladder, l2-3: DelH-F1a without DMSO, l4-5: DelH-F1a with DMSO, l6-7: DelH-F1b, l8:2log ladder, l9:pSB1C3 l10: pSB1C3-AraC-lacZ

The gel shows a lot unspecific bands for both DelH F1a samples.

=> Alter PCR conditions.

DelH F1b

  • The new primer arrived

PCR Conditions F1b.W4.A

Reagent DelH F1b
Expected length [Kb] 5
Template 1 µl D. acidovorans
Primer fw 10 µM DN07
Primer rev 10 µM DN02
Phusion Master Mix (2x) 25 µl
DMSO -
ddH2O 23 µl
Cycles Temperature [°C] Time [s]
1 98 10
16 98 1 s
62 5 s
72 2:00 min
1 72 2:30 min
1 4 inf

Result

Expected band: 5 Kb

Fig.4.1 PCR ofDelH-fragments and pSB1C3(loaded 20 µL)
l1:2log ladder, l2-3: DelH-F1a without DMSO, l4-5: DelH-F1a with DMSO, l6-7: DelH-F1b, l8:2log ladder, l9:pSB1C3 l10: pSB1C3-AraC-lacZ

The gel shows a lot unspecific bands for DelH F1b, but also the band at 5 Kb.

=> Band was cut and gel isolated.

Generation of Backbone pSB6A1-AraC-lacZ

Miniprep

  • From three different clones, 2ml of pSB6A1-AraC-lacZ (6) in LB Amp were minipreped and eluted in 30 µl ddH2O. Additionally, a glycerol stock was prepared.

Result

Sample Concentration [ng/µl]
8 - pSB6A1-AraC-lacZ (6) 63
9 - pSB6A1-AraC-lacZ (6) 61
10 - pSB6A1-AraC-lacZ (6) 62


PCR Conditions BB.W4.A

Reagent 8 - pSB6A1-AraC-lacZ (6) 9 - pSB6A1-AraC-lacZ (6)
Expected length [Kb] 7.382 7.382
Template 1 µl of 1:10 diluted 8 1 µl of 1:10 diluted 9
Primer 10 µM fw 0.5 µl
Primer 10 µM rev 0.5 µl
Phusion Master Mix (2x) 25 µl 25 µl
DMSO - -
ddH2O 23 µl 23 µl
Cycles Temperature [°C] Time [s]
1 98 10
16 98 1 s
66↓ 5 s
72 3:00 min
14 98 1 s
63 5 s
72 3:00 min
1 72 5:00 min
1 4 inf

Results

Expected band: 7.4 Kb

Fig.4.2 PCR of BB pSB6A1-AraC-lacZ (loaded 50 µL)
l1:2log ladder, l2-3: pSB6A1-AraC-lacZ (8),l4:2log ladder, l5-6: pSB6A1-AraC-lacZ (10)
no band visible

The gel shows no bands.

=> Perform test restriction to test identity of plasmid.

Restriction Digest A

  • Digest with PstI & XbaI of samples 8, 9 and 10 for checking identity of the construct
Reagent Amount [µl]
DNA 10
Enzymes 1.5 each
Buffer NEB 3 (10x) 5
BSA (10x) 5
ddH2O 27
  • Incubated for 1 h at 37°C

Result

Expected bands: 3360 and 4022 bp

Fig.4.3 PCR of BB pSB6A1-AraC-lacZ (loaded 50 µL)
l1:2log ladder, l2: pSB6A1-AraC-lacZ (8), l3: pSB6A1-AraC-lacZ (9), l4: pSB6A1-AraC-lacZ (10),l5:2log ladder, l6: pSB6A1-AraC-lacZ (8) digested with XbaI & PstI, l7: pSB6A1-AraC-lacZ (9) digested with XbaI & PstI, l8: pSB6A1-AraC-lacZ (10)digested with XbaI & PstI, l9:2log ladder, l10: pSB6A1-AraC-lacZ (8) 1:10 diluted, l11: pSB6A1-AraC-lacZ (10) 1:10 diluted, l12: pSB1C3

The gel shows a band at 5 Kb.

=> Colonies 8, 9 and 10 are not the desired construct.

Restriction Digest B

  • Digest with KpnI of samples 8, 9, 10 for checking length of the construct and KpnI and BamHI to check for identity.
Reagent Amount [µl]
pSB6A1-AraC-lacZ (8, 9, 10) 5
Enzymes 1.5 KpnI
Buffer NEB 3 (10x) 5
BSA (10x) 5
ddH2O 33,5
Expected bands 7.381 bp
Reagent Amount [µl]
pSB6A1-AraC-lacZ (8, 9, 10) 5
Enzymes 1.5 KpnI & 1,5 BamHI
Buffer NEB 3(10x) 5
BSA (10x) 5
ddH2O 32
Expected bands 4.607 bp & 2.781 bp

Result

  • Expected bands: 7.381 bp and 4.607 bp & 2.781 bp


Fig.4.4 BB pSB6A1-AraC-lacZ test digested (loaded 50 µL)
l1:2log ladder, l2: pSB13-AraC-lacZ (8), l3: pSB6A1, l4-6: pSB6A1-AraC-lacZ digested with BpsI, l7:2log ladder, l8: pSB13-AraC-lacZ (8), l9: pSB6A1, l10-12: pSB6A1-AraC-lacZ digested with BpsI & BamHI
=> Colonies 8, 9 and 10 are not desired construct. Pick new colony from pSB1C3-AraC(6)-lacZ and test digest.

Miniprep

A new colony was picked from pSB1C3-AraC(6)-lacZ plate and a mini prep was performed.

Result

pSB6A1-AraC(6)-lacZ construct : 94 [ng/µl]

Test Restriction Digest

The new miniprep as well as one of the wrong pSB6A1-AraC(6)-lacZ ones were restriction digested with EcoRI-HF and PstI.

Component Amount [µl]
DNA 5
BSA (10x) 5
NEB2 buffer (10x) 5
Enzymes (EcoRI-HF and PstI) 1.5 each
ddH2O 32
  • Incubated 1 h at 37°C

Result

Expected bands:  ? Kb

Fig.4.5 Test digest of pSB1C3-AraC-lacZ with EcoRI & PstI (loaded 20 µL)
l1:2log ladder, l2: pSB1C3-AraC-lacZ

The gel showed again these three bands (2.070 and 3.360 and ~4 Kb)

=> Colonies 8, 9 and 10 are not the desired construct.

Generation of Backbone pSB1C3-AraC-lacZ

Test Restriction Digest

In order find out why pSB6A1-AraC-lacZ shows wrong restriction pattern, parental pSB1C3-AraC-lacZ was restriction digested using KpnI as well as KpnI & BamHI.

Reagent Amount [µl]
pSB1C3-AraC-lacZ 5
Enzymes 1.5 KpnI
Buffer NEB 3(10x) 5
BSA (10x) 5
ddH2O 33.5
Expected band 5,436 bp (linerized)
Reagent Amount [µl]
pSB1C3-AraC-lacZ 5
Enzymes 1.5 KpnI & 1.5 BamHI
Buffer NEB 3(10x) 5
BSA (10x) 5
ddH2O 32
Expected bands 4.906 bp & 530 bp

Result

Expected bands: 5.436 bp and 4.906 bp & 530 bp

Fig.4.4 BB pSB6A1-AraC-lacZ test digested (loaded 50 µL)
l1:2log ladder, l2: pSB13-AraC-lacZ (8), l3: pSB6A1, l4-6: pSB6A1-AraC-lacZ digested with BpsI, l7:2log ladder, l8: pSB13-AraC-lacZ (8), l9: pSB6A1, l10-12: pSB6A1-AraC-lacZ digested with BpsI & BamHI
=> pSB1C3-AraC(6)-lacZ is not desired construct. Pick new colony and test digest.

Miniprep

A new colony was picked from pSB1C3-AraC(6)-lacZ plate and a mini prep was performed.

Result

pSB1C3-AraC(6)-lacZ construct : 203 [ng/µl]

Test Restriction Digest

The new miniprep as well as one of the wrong pSB6A1-AraC(6)-lacZ ones were restriction digested with EcoRI-HF and PstI.

Component Amount [µl]
DNA 3
BSA (10x)5
NEB2 buffer (10x) 5
Enzymes (EcoRI-HF and PstI) 1.5 each
ddH2O 34
  • Incubated 1 h at 37°C

Result

Expected band:  ? Kb

Fig.4.6 BB digested with EcoRI & PstI (loaded 20 µL)
l1:2log ladder, l2: digested pSB1C3-AraC-lacZ, l3: digested pSB6A1-AraC-lacZ

Gel showed again the three bands (2070 and 3360 and ~4 Kb).

=> pSB1C3-AraC(6)-lacZ is not desired construct. Start assembly all over again.

Test Restriction Digest pSB6A1-J04450

pSB6A1-J04450 was test digested to check for identity.

Reagent Amount [µl]
pSB6A1-J04450 10
Enzymes 1.5 KpnI
Buffer NEB 3 (10x) 5
BSA (10x) 5
ddH2O 28.5
Expected band 5,091 bp - Linearized
Reagent Amount [µl]
pSB6A1-J04450 10
Enzymes 1.5 KpnI & 1.5 BamHI
Buffer NEB 3 (10x) 5
BSA (10x) 5
ddH2O 27
Expected bands 4,906 bp & 530 bp

Result

Fig.4.4 BB pSB6A1-AraC-lacZ test digested (loaded 50 µL)
l1:2log ladder, l2: pSB13-AraC-lacZ (8), l3: pSB6A1, l4-6: pSB6A1-AraC-lacZ digested with BpsI, l7:2log ladder, l8: pSB13-AraC-lacZ (8), l9: pSB6A1, l10-12: pSB6A1-AraC-lacZ digested with BpsI & BamHI
Sample Expected bands with KpnI Bands on gel with KpnI Expected bands with KpnI & BamHI Bands on gel with KpnI & BamHI
pSB1C3-AraC-lacZ 5.436 Kb (linerized) 10 Kb 4.906 Kb & 530 Kb no band
pSB6A1-J04450 5.091 Kb (linearized) no band 5.091 Kb (linearized) no band
pSB6A1-AraC-lacZ (8, 9, 10) 7.388 Kb (linearized) 6 Kb & 10 Kb 4.607 Kb & 2.781 Kb 6Kb & 10 Kb


This means:

  • The chloramphenicol backbone has the right length.
  • The digested chloramphenicol backbone is also ok.
  • The digested fragment pSB6A1-AraC-lacZ (with PstI & EcoRI-HF) showed the 2 expected bands at 2,070 bp and 3,360 bp, but another band around ~4 Kb.
=> Something is not ok with this ligated construct and we have to start all over again.


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