Team:Heidelberg/Templates/DelH week 3

From 2013.igem.org

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[[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 ]]
[[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 ]]
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Different unspecific bands were observed.
Different unspecific bands were observed.
:=> Test digest to confirm identity.
:=> Test digest to confirm identity.
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<br/>
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<div style="clear:both"></div>
====Test Restriction Digest====
====Test Restriction Digest====
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[[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 ]]
[[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 ]]
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There is only an unspecific band at the second PCR of F1 with 2.5 µl DMSO.
There is only an unspecific band at the second PCR of F1 with 2.5 µl DMSO.
:=> Repeat using DMSO and altered PCR program.
:=> Repeat using DMSO and altered PCR program.
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<div style="clear:both"></div>
====PCR Conditions F1.W3.C====
====PCR Conditions F1.W3.C====
{| class="wikitable" style="float:left; margin-right:1em"
{| class="wikitable" style="float:left; margin-right:1em"
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[[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  ]]
[[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  ]]
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There was no band visible.
There was no band visible.
:=> Repeat using DMSO and altered PCR program.
:=> Repeat using DMSO and altered PCR program.
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<br/>
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<div style="clear:both"></div>
===Amplification of DelH F2===
===Amplification of DelH F2===
====Gel Purification====
====Gel Purification====
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[[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 ]]
[[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 ]]
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Specific band was observed.
Specific band was observed.
:=> Test digest to confirm identity.
:=> Test digest to confirm identity.
<br/>
<br/>
-
 
+
<div style="clear:both"></div>
====Test Restriction Digest====
====Test Restriction Digest====
{| class="wikitable"
{| class="wikitable"
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<br/>
<br/>
[[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 ]]
[[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 ]]
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<br/>
+
 
There is no band visible.
There is no band visible.
:=> Repeat using altered PCR program.
:=> Repeat using altered PCR program.
<br/>
<br/>
-
 
+
<div style="clear:both"></div>
====PCR Conditions F2.W3.B====
====PCR Conditions F2.W3.B====
{| class="wikitable" style="float:left; margin-right:1em"
{| class="wikitable" style="float:left; margin-right:1em"
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[[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 ]]
[[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 ]]
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<div style="clear:both"></div>
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There was a specific band at 8 Kb
There was a specific band at 8 Kb
:=> Fragment was cut and gel extracted.
:=> Fragment was cut and gel extracted.
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<br/>
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<div style="clear:both"></div>
===Generation of Backbone pSB6A1-AraC-lacZ===
===Generation of Backbone pSB6A1-AraC-lacZ===
====Miniprep of Amplified Parts====
====Miniprep of Amplified Parts====
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[[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 ]]
[[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 ]]
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<div style="clear:both"></div>
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There was no fragments visible.
There was no fragments visible.
:=> Is picked coloniy negative or did entire ligation not work out?
:=> Is picked coloniy negative or did entire ligation not work out?
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<br/>
 +
<div style="clear:both"></div>

Latest revision as of 11:37, 23 October 2013

Contents

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?


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