Team:Heidelberg/Templates/DelH week5

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Contents

27-05 - 02-06-13

Generation of pSB6A1

Miniprep

  • Grow pSB6A1 from glycerol stock in 2 ml LB Amp ON at 37°C.
  • Miniprep using miniprep kit from Qiagen and dilute in 30 µl ddH2O.

Result

Concentration 69.5 ng/µl

Digest

  • The pSB6A1 was generated from the parts registry and cut with EcoRI & PstI
Template DNA BSA NEBuffer2 Enzymes ddH2O Total amount
15 µl (69.5 ng/µl) 5 µl 5 µl 2x 1.5 µl EcoRI-HF & PstI 22 µl 50 µl
The digest was incubated 45 min at 37°C.

Result

Expected band: 3985 bp (pSB6A1) and at 1106 bp (insert=J04450)

Fig.5.1 Generation of pSB6A1 for Backbone generation (loaded 50 µL of PCR)
l1:2-log ladder, l2-3:digested pSB6A1
expected band at 4 Kb was cut out
=> Expected bands are present. Backbone pSB6A1 was cut out and gel purified (c=4 ng/µl).

Generation of AraC Fragment

Gel Extraction

Gel extraction was performed with the gel extraction kit from Qiagen of gel slice (cut 24-05) and was eluted in 30 µl.

Result

The measured concentrations are shown in the table below and was stored at -20°C.

Sample Concentration [ng/µl]
AraC 22.5

Generation of lacZ Fragment

Gel Extraction

Gel extraction was performed with the gel extraction kit from Qiagen of gel slice (cut 24-05) and was eluted in 30 µl.

Result

The measured concentrations are shown in the table below and was stored at -20°C.

Sample Concentration [ng/µl]
lacZ 42.5

Generation of Backbone pSB6A1-AraC-lacZ

Ligation of pSB6A1 with AraC-lacZ

A ligation was performed of fragments for 1 h at RT.

Sample Concentration [ng/µl] Amount for ligation [µl]
pSB6A1 4 12.5
AraC-lacZ 22.5 3.5
T4-ligase - 1
Buffer - 2
ddH2O - 1
Final volume - 20
  • The ligase was inactivated by heat shock for 5 min at 70°C.

Transformation

  • Afterwards, a transformation was performed with 15 µl of the ligation reaction in competent TOP10.
  • Cells were streaked on LB Amp plates and incubated ON at 37°C.

Induction

  • The following day, 5 white colonies were picked (because the red ones still have the mRFP insert J04450) and were incubated in 2 ml LB Amp with 20 µl Arabinose (10%) and 100 µl X-Gal (20 ng/µl).

Result

2 of the colonies turned blue

=> Perform miniprep.

Miniprep

Miniprep was performed according to Qiagen's instructions.

Result

Sample Concentration [ng/µl]
pSB6A1-AraC-lacZ 99


DelH F1a

PCR Conditions F1a.W5.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
62 5
72 2:00 min
1 72 2:30 min
1 4 inf

Result

Expected band: 5 Kb

Fig.5.2 PCR of DelH fragment F1a, F1b & F2(loaded 50 µL of PCR)
l1:2-log ladder, l3-4:F1a,l5-6:F1b,l7-8:F2

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

=> Fragment was cut and gel purified.


DelH F1b

PCR Conditions F1b.W5.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
68 5
72 2:00 min
1 72 2:30 min
1 4 inf

Result

Expected band: 5 Kb

Fig.5.2 PCR of DelH fragment F1a, F1b & F2(loaded 50 µL of PCR)
l1:2-log ladder, l3-4:F1a,l5-6:F1b,l7-8:F2

Gel shows one band at the expected length of 5 Kb, but also other side bands below and a huge, bright band at 2 Kb.

=> Band was cut and gel isolated.


DelH F2

PCR Conditions F2.W5.A

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.5.2 PCR of DelH fragment F1a, F1b & F2(loaded 50 µL of PCR)
l1:2-log ladder, l3-4:F1a,l5-6:F1b,l7-8:F2

There was a specific band at 8 Kb.

=> Fragment was cut and gel extracted.


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