Team:Heidelberg/Templates/DelH week4

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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.

Team:Heidelberg/Templates/DelH week4

From 2013.igem.org

Contents

20-05 - 26-05-13

DelH Fragment F1a

PCR Conditions F1a.W4.A

Results

DelH F1b

PCR Conditions F1b.W4.A

Result

Generation of Backbone pSB6A1-AraC-lacZ

Miniprep

Result

PCR Conditions BB.W4.A

Results

Restriction Digest A

Result

Restriction Digest B

Result

Miniprep

Result

Test Restriction Digest

Result

Generation of Backbone pSB1C3-AraC-lacZ

Test Restriction Digest

Result

Miniprep

Result

Test Restriction Digest

Result

Test Restriction Digest pSB6A1-J04450

Result