Team:Heidelberg/Templates/DelH week 3

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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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