Team:Heidelberg/Templates/DelH week 3

From 2013.igem.org

(Difference between revisions)

Revision as of 20:46, 1 October 2013

13-05 - 19-05-13

Amplification of DelH F1

Gel Purification

45 µl of DelH F1 PCR (08-05) product were run on a 0,8% agarose gel (1 h with 135V). Gel extraction was performed using gel extraction kit of Qiagen, and diluted in 25 µl ddH₂O.

Result

Sample	Concentration [ng/µl]
DelH F1	7

=> Because the concentration is so low, a re-PCR of the PCR product (08-05) is going to be done.

re-PCR Conditions F1.W3.A

Reagent	DelH F1
Expected length [Kb]	10
Template	1 µl 1:10 gel purified F1
Primer 10 µM fw	0.5 µl DelH_f1_PacI_fw
Primer 10 µM rev	0.5 µl DelH_f1_SalI_rev
Phusion Master Mix (2x)	25 µl
DMSO	2.5 µl
ddH₂O	20.5 µl

Cycles	Temperature [°C]	Time [s]
1	98	10
30	98	1
	66	5
	72	3:30 min
72	3:30 min
1	4	inf

Result

Expected length: 10 Kb

Fig.3.1 PCR of DelH fragment 1 (loaded 20 µL)
l1:fragment 1 of DelH, l2: 2log ladder

Different unspecific bands were observed.

=> Test digest to confirm identity.

Test Restriction Digest

PCR product of DelH-F1	BSA	NEBuffer 4	Enzymes	ddH₂O	Total amount
18 µl	5 µl	5 µl	2x 1.5 µl SalI-HF & PacI	19 µl	50 µl

Afterwards, purification with the nucleotide removal kit was performed, but due to using wrong column, there was no product left.

=> Repeat amplification of DelH F1.

PCR Conditions F1.W3.B

Reagent	DelH F1	DelH F1	DelH F1
Expected length [Kb]	10	10	10
Template	Picked colony	Picked colony	Picked colony
Primer 10 µM fw	0.5 µl DelH_f1_PacI_fw	0.5 µl DelH_f1_PacI_fw	0.5 µl DelH_f1_PacI_fw
Primer 10 µM rev	0.5 µl DelH_f1_SalI_rev	0.5 µl DelH_f1_SalI_rev	0.5 µl DelH_f1_SalI_rev
Phusion Master Mix (2x)	25 µl	25 µl	25 µl
DMSO	1.5 µl	2.5 µl	5 µl
ddH₂O	21.5 µl	20.5 µl	18 µl

Cycles	Temperature [°C]	Time [s]
1	98	10
30	98	1 s
	66	5 s
	72	3:30 min
1	72	3:30 min
1	4	inf

Result

Expected length: 10 Kb

Fig.3.2 PCR of DelH fragment 1 (loaded 20 µL)
l1:2log ladder, l2-4: fragment 1 of DelH, l5: fragment 2 of DelH

There is only an unspecific band at the second PCR of F1 with 2.5 µl DMSO.

=> Repeat using DMSO and altered PCR program.

PCR Conditions F1.W3.C

Reagent	DelH F1	DelH F1
Expected length [Kb]	10	10
Template	1 µl glycerol stock	1 µl glycerol stock
Primer 10 µM fw	0.5 µl DelH_f1_PacI_fw	0.5 µl DelH_f1_PacI_fw
Primer 10 µM rev	0.5 µl DelH_f1_SalI_rev	0.5 µl DelH_f1_SalI_rev
Phusion Master Mix (2x)	25 µl	25 µl
DMSO	2.5 µl	2.5 µl
ddH₂O	20.5 µl	20.5 µl

Cycles	Temperature [°C]	Time [s]
1	98	10
30	98	1 s
	66	5 s
	72	3:30 min
1	72	3:30 min
1	4	inf

Result

Expected length: 10 Kb

Fig.3.3 PCR of DelH fragment 1 (loaded 20 µL)
l1:2log ladder, l2: fragment 1 of DelH, l3: fragment 2 of DelH, l4: fragment 1 of DelH, l5: fragment 2 of DelH

There was no band visible.

=> Repeat using DMSO and altered PCR program.

Amplification of DelH F2

Gel Purification

45 µl of DelH F1 PCR (07-05) product were run on a 0,8% agarose gel (1 h with 135V). Gel extraction was performed using gel extraction kit of Qiagen, and diluted in 25 µl ddH₂O.

Result

Sample	Concentration [ng/µl]
DelH F2	6

=> Because the concentration is so low, a re-PCR of the PCR product (07-05) is going to be done.

Re-PCR Conditions F2.W3.A

Reagent	DelH-fragment2
Expected length [Kb]	8
Template	1 µl 1:10 gel purified F2
Primer 10 µM fw	0.5 µl DelH_f2_SalI_fw
Primer 10 µM rev	0.5 µl DelH_f2_KpnI_rev
Phusion Master Mix (2x)	25 µl
DMSO	2.5 µl
ddH₂O	20.5 µl

Cycles	Temperature [°C]	Time [s]
1	98	10
30	98	1 s
	66	5 s
	72	3:30 min
72	3:30 min
1	4	inf

Result

Expected length: 8 Kb

Fig.3.4 PCR of DelH fragment 1 (loaded 20 µL)
l1:2log ladder, l2: fragment 2 of DelH

Specific band was observed.

=> Test digest to confirm identity.

Test Restriction Digest

PCR product of DelH-F1	BSA	NEBuffer 4	Enzymes	ddH₂O	Total amount
18 µl	5 µl	5 µl	2x 1.5 µl SalI-HF & KpnI-HF	19 µl	50 µl

Afterwards, a purification with the nucleotide removal kit was performed, but due to using wrong column, there was no product left.

=> Repeat amplification of DelH F2.

PCR Conditions F2.W3.B

Reagent	DelH F2
Expected length [Kb]	8
Template	Picked colony
Primer 10 µM fw	0.5 µl DelH_f2_SalI_fw
Primer 10 µM rev	0.5 µl DelH_f2_KpnI_rev
Phusion Master Mix (2x)	25 µl
DMSO	0 µl
ddH₂O	23 µl

Cycles	Temperature [°C]	Time [s]
1	98	10
30	98	1
	66	5
	72	2:45 min
1	72	2:45 min
1	4	inf

Result

Expected length: 8 Kb

Fig.3.2 PCR of DelH fragment 1 (loaded 20 µL)
l1:2log ladder, l2: fragment 1 of DelH, l3: fragment 2 of DelH

There is no band visible.

=> Repeat using altered PCR program.

PCR Conditions F2.W3.B

Reagent	DelH F2
Expected length [Kb]	8
Template	1 µl glycerol stock
Primer 10 µM fw	0.5 µl DelH_f2_SalI_fw
Primer 10 µM rev	0.5 µl DelH_f2_KpnI_rev
Phusion Master Mix (2x)	25 µl
DMSO	0 µl
ddH₂O	23 µl

Cycles	Temperature [°C]	Time [s]
1	98	10
30	98	1
	66	5
	72	2:45 min
1	72	2:45 min
1	4	inf

Result

Expected length: 8 Kb

Fig.3.3 PCR of DelH fragment 1 (loaded 20 µL)
l1:2log ladder, l2: fragment 1 of DelH, l3: fragment 2 of DelH

There was a specific band at 8 Kb

=> Fragment was cut and gel extracted.

Generation of Backbone pSB6A1-AraC-lacZ

Miniprep of Amplified Parts

DH10ß containing I13453 and K20600 (for AraC) and backbone pSB1AK3 (lacZ) were grown ON at 37°C and minipreped.

Result

Sample	Concentration [ng/µl]
lacZ	42.5
AraC (5)	26.5
AraC (6)	34.5

Restriction Digest

AraC was cut with EcoRI-HF and SpeI buffered in NEB4
LacZ was cut with XbaI and PstI buffered in NEB3

Reagent	Amount [µl]
DNA	22.5
Enzymes	1.5 each
Buffer (10x)	5
BSA (10x)	5
ddH₂O	14.5

Incubated for 1h at 37°C
Purified with Qiagen Kit and diluted in 20 µl ddH₂O

Result

Sample	Concentration [ng/µl]
lacZ	33.5
AraC (5)	19.5
AraC (6)	25.5

Ligation of pSB6A1, AraC and lacZ

Reagent	Amount [µl]
pSB6A1	1.5
lacZ	4
AraC	3
Ligase	1.5
Buffer	2
ddH₂O	8.5

Incubated at RT for 45 min
Chemical transformation of competent DH10ß
Streaked on LB Amp plates
Incubation ON at 37°C

Miniprep of pSB6A1-AraC-lacZ

One colony each was picked and grown in 2 ml LB Amp ON
Cultures were minipreped.

Result

Sample	Concentration [ng/µl]
pSB6A1-AraC-lacZ (5)	57
pSB6A1-AraC-lacZ (6)	73

Restriction Digest of Backbone pSB6A1-AraC-lacZ

AraC-lacZ was cut from pSB6A1-AraC-lacZ using PstI & EcoRI-HF

Reagent	Amount [µl]
Miniprep DNA	10
Enzymes	1.5 each
Buffer NEB 2(10x)	5
BSA (10x)	5
ddH₂O	27

Incubated for 1 h at 37°C
Purified with Qiagen kit and diluted in 20 µl ddH₂O

Result

Sample	Concentration [ng/µl]
pSB6A1-AraC-lacZ (5) D+P	18
pSB6A1-AraC-lacZ (6) D+P	26

Ligation of AraC-lacZ with pSB1C3

Reagent	Amount [µl]
DNA of pSB1C3	10
DNA of AraC-lacZ (6)	5
Ligase	1
Buffer	2
ddH₂O	2

Incubated at RT for 50 min
Heat inactivation at 70°C for 5 min
10 µl were used for a chemical transformation in TOP10 and plated on LB Chlor
Incubation ON at 37°C

Conlony-PCR Conditions BB.W3.A

Reagent	psB1C3-AraC-lacZ (5)
Expected length [Kb]	?
Template	Picked colony of psB1C3-AraC-lacZ (5)
Primer 10 µM fw	0.5 µl
Primer 10 µM rev	0.5 µl
Phusion Master Mix (2x)	25 µl
DMSO	-
ddH₂O	24 µl

Cycles	Temperature [°C]	Time [s]
1	98	10
30	98	1
	66	5
	72	1:30 min
1	72	1:30 min
1	4	inf

Result

Expected length:

Fig.3.5 PCR of BB psB1C3-AraC-lacZ(loaded 20 µL)
l1:2log ladder, l2: psB1C3-AraC-lacZ
no yield

There was no fragments visible.

=> Is picked coloniy negative or did entire ligation not work out?

@@ Line 52: / Line 52: @@
 Expected length: 10 Kb
 <br/>
-[[File:20130513 2log F1.png|200px|thumb|right|'''Fig.3.1''' PCR of DelH fragment 1 (loaded 20 µL) <br> ''l1:''fragment 1 of DelH, ''l2:'' 2log ladder ]]
+[[File:Heidelberg_20130513 2log F1.png|200px|thumb|right|'''Fig.3.1''' PCR of DelH fragment 1 (loaded 20 µL) <br> ''l1:''fragment 1 of DelH, ''l2:'' 2log ladder ]]
 <br/>
 Different unspecific bands were observed.
@@ Line 111: / Line 111: @@
 Expected length: 10 Kb
 <br/>
-[[File:20130516 2log 3xF1 F2.png|200px|thumb|right|'''Fig.3.2''' PCR of DelH fragment 1 (loaded 20 µL) <br> ''l1:''2log ladder, ''l2-4:'' fragment 1 of DelH, ''l5:'' fragment 2 of DelH ]]
+[[File:Heidelberg_20130516 2log 3xF1 F2.png|200px|thumb|right|'''Fig.3.2''' PCR of DelH fragment 1 (loaded 20 µL) <br> ''l1:''2log ladder, ''l2-4:'' fragment 1 of DelH, ''l5:'' fragment 2 of DelH ]]
 <br/>
 There is only an unspecific band at the second PCR of F1 with 2.5 µl DMSO.
@@ Line 156: / Line 156: @@
 Expected length: 10 Kb
 <br/>
-[[File:20130516 2log F1 F2 differentcycler.png|200px|thumb|right|'''Fig.3.3''' PCR of DelH fragment 1 (loaded 20 µL) <br> ''l1:''2log ladder, ''l2:'' fragment 1 of DelH, ''l3:'' fragment 2 of DelH,  ''l4:'' fragment 1 of DelH, ''l5:'' fragment 2 of DelH  ]]
+[[File:Heidelberg_20130516 2log F1 F2 differentcycler.png|200px|thumb|right|'''Fig.3.3''' PCR of DelH fragment 1 (loaded 20 µL) <br> ''l1:''2log ladder, ''l2:'' fragment 1 of DelH, ''l3:'' fragment 2 of DelH,  ''l4:'' fragment 1 of DelH, ''l5:'' fragment 2 of DelH  ]]
 <br/>
 There was no band visible.
@@ Line 213: / Line 213: @@
 Expected length: 8 Kb
 <br/>
-[[File:20130513 2log F2.png|200px|thumb|right|'''Fig.3.4''' PCR of DelH fragment 1 (loaded 20 µL) <br> ''l1:''2log ladder, ''l2:''  fragment 2 of DelH ]]
+[[File:Heidelberg_20130513 2log F2.png|200px|thumb|right|'''Fig.3.4''' PCR of DelH fragment 1 (loaded 20 µL) <br> ''l1:''2log ladder, ''l2:''  fragment 2 of DelH ]]
 <br/>
 Specific band was observed.
@@ Line 270: / Line 270: @@
 Expected length: 8 Kb
 <br/>
-[[File:20130516 2log 3xF1 F2.png|200px|thumb|right|'''Fig.3.2''' PCR of DelH fragment 1 (loaded 20 µL) <br> ''l1:''2log ladder, ''l2:'' fragment 1 of DelH, ''l3:'' fragment 2 of DelH ]]
+[[File:Heidelberg_20130516 2log 3xF1 F2.png|200px|thumb|right|'''Fig.3.2''' PCR of DelH fragment 1 (loaded 20 µL) <br> ''l1:''2log ladder, ''l2:'' fragment 1 of DelH, ''l3:'' fragment 2 of DelH ]]
 <br/>
 There is no band visible.
@@ Line 315: / Line 315: @@
 Expected length: 8 Kb
 <br/>
-[[File:20130516 2log F1 F2 differentcycler.png|200px|thumb|right|'''Fig.3.3''' PCR of DelH fragment 1 (loaded 20 µL) <br> ''l1:''2log ladder, ''l2:'' fragment 1 of DelH, ''l3:'' fragment 2 of DelH ]]
+[[File:Heidelberg_20130516 2log F1 F2 differentcycler.png|200px|thumb|right|'''Fig.3.3''' PCR of DelH fragment 1 (loaded 20 µL) <br> ''l1:''2log ladder, ''l2:'' fragment 1 of DelH, ''l3:'' fragment 2 of DelH ]]
 <div style="clear:both"></div>
 There was a specific band at 8 Kb
@@ Line 489: / Line 489: @@
 Expected length:
 <br/>
-[[File:20130515 2log AraC-lacZ-pSB1C3.png|200px|thumb|right|'''Fig.3.5''' PCR of BB psB1C3-AraC-lacZ(loaded 20 µL) <br> ''l1:''2log ladder, ''l2:'' psB1C3-AraC-lacZ <br/> no yield ]]
+[[File:Heidelberg_20130515 2log AraC-lacZ-pSB1C3.png|200px|thumb|right|'''Fig.3.5''' PCR of BB psB1C3-AraC-lacZ(loaded 20 µL) <br> ''l1:''2log ladder, ''l2:'' psB1C3-AraC-lacZ <br/> no yield ]]
 <div style="clear:both"></div>
 There was no fragments visible.
 :=> Is picked coloniy negative or did entire ligation not work out?
 <br/>

Team:Heidelberg/Templates/DelH week 3

From 2013.igem.org

Revision as of 20:46, 1 October 2013

Contents

13-05 - 19-05-13

Amplification of DelH F1

Gel Purification

Result

re-PCR Conditions F1.W3.A

Result

Test Restriction Digest

PCR Conditions F1.W3.B

Result

PCR Conditions F1.W3.C

Result

Amplification of DelH F2

Gel Purification

Result

Re-PCR Conditions F2.W3.A

Result

Test Restriction Digest

PCR Conditions F2.W3.B

Result

PCR Conditions F2.W3.B

Result

Generation of Backbone pSB6A1-AraC-lacZ

Miniprep of Amplified Parts

Result

Restriction Digest

Result

Ligation of pSB6A1, AraC and lacZ

Miniprep of pSB6A1-AraC-lacZ

Result

Restriction Digest of Backbone pSB6A1-AraC-lacZ

Result

Ligation of AraC-lacZ with pSB1C3

Conlony-PCR Conditions BB.W3.A

Result