Team:Heidelberg/Templates/DelH week12

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Contents

15-07 - 21-07-13

Preparation of Primers

  • All primers were 1:10 diluted
  • Overview on annealing temperatures
Primer short2 HM02 HM03 HM04 HM05 HM06 HM07 HM08 HM09 HM10
Annealing temperature [°C] 63 72.6 69.1 55.2 68.2 66.4 69.6 65.2 51.6 / 73.6 60.9 / 71.3


Preparation of fresh D. acidovorans

  • Liquid culture: 2x 5 ml ACM medium was inocculated with picked colonies of D. acidovorans from plate (made by DN)
  • Glycerol stock: was prepared from liquid culture


Amplification of DelH G1

PCR Conditions G1.W12.A

Reagent DelH G1 DelH G1
Template Fresh colony of plate (by DN) Fresh colony of plate (by DN)
Primer fw 10 µM short2 short2
Primer rev 10 µM HM04 HM04
Phusion Flash Ready Mix 10 µl 10 µl
ddH2O 7 µl 6 µl
DMSO - 1 µl
Cycles Temperature [°C] Time [s]
1 98 5
12 98 1
63 (touchdown -0.5°C) 5
72 3:30 min
18 98 1
63 5
72 3:30 min
1 72 10 min
1 4 inf

Result

Expected band: 8.961 Kb

Fig.12.1gel of PCR of DelH-fragments (loaded 20 µL)
l1:F1 without DMSO,l2:F1 with DMSO, l3:2log ladder, l4: F2 without DMSO, l5: F2 with DMSO


No bands visible.

=> Fragment 1 was not amplified. Next step is amplifying fragment 1 in two parts G1a & G1b.


Amplification of DelH G1a

PCR Conditions G1a.W12.A

Reagent DelH G1a DelH G1a
Template Fresh colony of plate (by DN) Fresh colony of plate (by DN)
Primer fw 10 µM short2 short2
Primer rev 10 µM HM02 HM02
Phusion Flash Ready Mix 10 µl 10 µl
ddH2O 7 µl 6 µl
DMSO - 1 µl
Cycles Temperature [°C] Time [s]
1 98 5
12 98 1
63 (touchdown -0.5°C) 5
72 3:30 min
18 98 1
64 5
72 3:30 min
1 72 10 min
1 4 inf

Result

Expected Band: 4.309 Kb

Fig.12.3gel of PCR of DelH-fragments (loaded 20 µL)
l1:F1a without DMSO,l2:F1a with DMSO, l3:F16 without DMSO at an anealing temperature 64°C,l5:F16 with DMSO at an anealing temperature 64°C,l6:1b-57 (without DMSO), l7: 1b-57 (with DMSO),l9 2 loga ladder,l10 Mediprep A of BB,l11 Mediprep B of BBF1a with DMSO
Fig.12.2gel of PCR of DelH-fragments (loaded 20 µL)
l1:F1a without DMSO,l2:F1a with DMSO, l3:F16 without DMSO at an anealing temperature 64°C,l5:F16 with DMSO at an anealing temperature 64°C,l6:1b-57 (without DMSO), l7: 1b-57 (with DMSO),l9 2 loga ladder,l10 Mediprep A of BB,l11 Mediprep B of BBF1a with DMSO

PCR using DMSO looks well.

=> Fragment was cut and gel extracted.


Amplification of DelH G1b

PCR Conditions G1b.W12.A and B

Reagent DelH G1b DelH G1b
Template Fresh colony of plate (by DN) Fresh colony of plate (by DN)
Primer fw 10 µM HM03 HM03
Primer rev 10 µM HM04 HM04
Phusion Flash Ready Mix 10 µl 10 µl
ddH2O 7 µl 6 µl
DMSO - 1 µl
Cycles Temperature A [°C] Time [s] Cycles Temperature B [°C] Time [s]
1 98 5 1 98 5
30 98 1 30 98 1
64 5 57 5
72 2:15 min 72 2:15 min
1 72 7 min 1 72 7 min
1 4 inf 1 4 inf

Result

Expected Band: 4.711 Kb

Fig.12.3gel of PCR of DelH-fragments (loaded 20 µL)
l1:F1a without DMSO,l2:F1a with DMSO, l3:F16 without DMSO at an anealing temperature 64°C,l5:F16 with DMSO at an anealing temperature 64°C,l6:1b-57 (without DMSO), l7: 1b-57 (with DMSO),l9 2 loga ladder,l10 Mediprep A of BB,l11 Mediprep B of BBF1a with DMSO
Fig.12.2gel of PCR of DelH-fragments (loaded 20 µL)
l1:F1a without DMSO,l2:F1a with DMSO, l3:F16 without DMSO at an anealing temperature 64°C,l5:F16 with DMSO at an anealing temperature 64°C,l6:1b-57 (without DMSO), l7: 1b-57 (with DMSO),l9 2 loga ladder,l10 Mediprep A of BB,l11 Mediprep B of BBF1a with DMSO


PCR using DMSO at an annealing temperature od 57°C looks fine.

=> Fragment was cut and gel extracted.


Amplification of DelH G2

PCR Conditions G2.W12.A

Reagent DelH G2 DelH G2
Template Fresh colony of plate (by DN) Fresh colony of plate (by DN)
Primer fw 10 µM 2 µl HM05 2 µl HM05
Primer rev 10 µM 2 µl HM08 2 µl HM08
Phusion Flash Ready Mix 10 µl 10 µl
ddH2O 8 µl 7 µl
DMSO - 1 µl
Cycles Temperature [°C] Time [s]
1 98 5
12 98 1
68 (touchdown -0.5°C) 5
72 3:30 min
18 98 1
67 5
72 3:30 min
1 72 10 min
1 4 inf

Result

Expected band: 9.611 Kb

Fig.12.1gel of PCR of DelH-fragments (loaded 20 µL)
l1:F1 without DMSO,l2:F1 with DMSO, l3:2 log ladder,l4:F2 (without DMSO), l5: F2 (with DMSO)

Expected band is visible.

=> Fragment G2 was amplified with DMSO. Band was cut and gel extracted.


PCR Conditions G2.W12.B

Reagent DelH G2
Template Fresh colony of plate (by DN)
Primer fw 10 µM 2.5 µl HM05
Primer rev 10 µM 2.5 µl HM08
Phusion Flash Ready Mix 25 µl
ddH2O 17.5 µl
DMSO 2.5 µl
Cycles Temperature [°C] Time [s]
1 98 5
12 98 1
68 (touchdown -0.5°C) 5
72 3:30 min
18 98 1
67 5
72 3:30 min
1 72 10 min
1 4 inf

Result

Expected band: 9.611 Kb

Fig.12.4gel of PCR of DelH-fragments (loaded 20 µL)
l1:PCR of Indigoidine,l2:2 log ladder, l3-4:G1b
l3-4 showed expected band at 9.6 Kb = was cut out

Gel shows nice band at expected length.

=> G2 was also amplified in 50 µl and was cut and gel extracted.


Amplification of DelH G0

PCR Conditions G0.W12.A

Reagent DelH G0
Template 1 µl of glycerol stock
Expected length [Kb] 18.521
Primer fw 10 µM 2.5 µl short2
Primer rev 10 µM 2.5 µl HM08
Phusion Flash Ready Mix 10 µl
ddH2O 5 µl
DMSO 1 µl
Cycles Temperature [°C] Time [s]
1 98 5
30 98 1
65 5
72 3:30 min
1 72 10 min
1 4 inf

Result

Expected band: 18.521 Kb

Fig.12.6gel of PCR of DelH-fragments (loaded 20 µL)
l1-4:DelH G0-G3 (18 Kb),l5:2 log ladder
Fig.12.5gel of PCR of DelH-fragments (loaded 20 µL)
l1-4:DelH G0-G3 (18 Kb),l5:2 log ladder


Gel shows expected band at ~18 Kb.

=> Fragment G0 was cut and gel isolated. Repeat PCR to increase yield.

Result

Three 20 µl reactions were performed to increase yield of fragment G0.
Expected band: 18.521 Kb

Fig.12.8gel of PCR of DelH-fragments (loaded 20 µL)
l1-4:DelH G0-G3 (18 Kb),l5:2 log ladder
Fig.12.7gel of PCR of DelH-fragments (loaded 20 µL)
l1-4:DelH G0-G3 (18 Kb),l5:2 log ladder

Gel shows expected band at ~18 Kb.

=> Fragment G0 was cut and gel isolated.


Amplification of DelH G1/2a

PCR Conditions G1/2a.W12.A

Reagent DelH G1/2a
Template 1 µl of glycerol stock
Expected length [Kb] 13.083
Primer fw 10 µM 2.5 µl short2
Primer rev 10 µM 2.5 µl HM06
Phusion Flash Ready Mix 10 µl
ddH2O 5 µl
DMSO 1 µl
Cycles Temperature [°C] Time [s]
1 98 5
30 98 1
65 5
72 4:45 min
1 72 10 min
1 4 inf

Result

Expected band: 13.083 Kb

Fig.12.6gel of PCR of DelH-fragments (loaded 20 µL)
l1-4:DelH G0-G3 (18 Kb),l5:2 log ladder
Fig.12.5gel of PCR of DelH-fragments (loaded 20 µL)
l1-4:DelH G0-G3 (18 Kb),l5:2 log ladder


Gel shows expected band at ~13 Kb.

=> Fragment G1/2a was cut and gel isolated.


Amplification of DelH G1b/2

PCR Conditions G1b/2.W12.A

Reagent DelH G1b/2
Template 1 µl of glycerol stock
Expected length [Kb] 14.271
Primer fw 10 µM 2.5 µl HM03
Primer rev 10 µM 2.5 µl HM08
Phusion Flash Ready Mix 10 µl
ddH2O 5 µl
DMSO 1 µl
Cycles Temperature [°C] Time [s]
1 98 5
30 98 1
65 5
72 3:30 min
1 72 10 min
1 4 inf

Result

Expected band: 14.271 Kb

Fig.12.6gel of PCR of DelH-fragments (loaded 20 µL)
l1-4:DelH G0-G3 (18 Kb),l5:2 log ladder
Fig.12.5gel of PCR of DelH-fragments (loaded 20 µL)
l1-4:DelH G0-G3 (18 Kb),l5:2 log ladder


Gel shows expected band at ~14 Kb.

=> Fragment G1b/2 was cut and gel isolated.


Amplification of DelH G1b/2a

PCR Conditions G1b/2a.W12.A

Reagent DelH G1b/2a
Template 1 µl of glycerol stock
Expected length [Kb] 14.271
Primer fw 10 µM 2.5 µl HM03
Primer rev 10 µM 2.5 µl HM06
Phusion Flash Ready Mix 10 µl
ddH2O 5 µl
DMSO 1 µl
Cycles Temperature [°C] Time [s]
1 98 5
30 98 1
65 5
72 3:30 min
1 72 10 min
1 4 inf

Result

Expected band: 14.271 Kb

Fig.12.6gel of PCR of DelH-fragments (loaded 20 µL)
l1-4:DelH G0-G3 (18 Kb),l5:2 log ladder
Fig.12.5gel of PCR of DelH-fragments (loaded 20 µL)
l1-4:DelH G0-G3 (18 Kb),l5:2 log ladder


Gel shows multiple bands, it is not clear, which one is correct.

=> Optimize temperatures.


Amplification of DelH G2b

PCR Conditions G2b.W12.A

Reagent DelH G2b
Template 1 µl of glycerol stock
Expected length [Kb] 5
Primer fw 10 µM 2.5 µl HM07
Primer rev 10 µM 2.5 µl HM08
Phusion Flash Ready Mix 10 µl
ddH2O 5 µl
DMSO 1 µl
Cycles Temperature [°C] Time [s]
1 98 5
30 98 1
68 5
72 2:00 min
1 72 5 min
1 4 inf

Result

Expected band: 5 Kb

Fig.12.8gel of PCR of DelH-fragments and restriction digest (loaded 20 µL)
l1-3:DelH G0(18 Kb),l5:1 Kb+ ladder, l5:Midiprep A digested with PstI & BamHI,l6:Midiprep A digested with SalI & BamHI, l7:Midiprep B digested with PstI & BamHI,l8:Midiprep B digested with SalI & BamHI, l9: 2 log ladder, l10: fragment of DelH - G3b
Fig.12.7gel of PCR of DelH-fragments and restriction digest (loaded 20 µL)
l1-3:DelH G0(18 Kb),l5:1 Kb+ ladder, l5:Midiprep A digested with PstI & BamHI,l6:Midiprep A digested with SalI & BamHI, l7:Midiprep B digested with PstI & BamHI,l8:Midiprep B digested with SalI & BamHI, l9: 2 log ladder, l10: fragment of DelH - G3b


Amplification of Backbone pSB6A1-AraC-lacZ

Test Restriction Digest of Midiprep

Midiprep A A B B
Enzymes BamHI & Pst BamHI & SalI BamHI & PstI BamHI & SalI
Wanted fragments [Kb] 3.317 & 4.071 3.794 & 3.594 3.317 & 4.071 3.794 & 3.594
Fragments present 1 band at ~6-7 Kb 3 bands: one bright one at ~4-5 Kb and two
smaller ones at ~6 Kb and ~10 Kb 		1 band at ~6-7 Kb 3 bands: one bright one at ~4-5 Kb and two
smaller ones at ~6 Kb and ~10 Kb
Fig.12.8gel of PCR of DelH-fragments and restriction digest (loaded 20 µL)
l1-3:DelH G0(18 Kb),l5:1 Kb+ ladder, l5:Midiprep A digested with PstI & BamHI,l6:Midiprep A digested with SalI & BamHI, l7:Midiprep B digested with PstI & BamHI,l8:Midiprep B digested with SalI & BamHI, l9: 2 log ladder, l10: fragment of DelH - G3b
Fig.12.7gel of PCR of DelH-fragments and restriction digest (loaded 20 µL)
l1-3:DelH G0(18 Kb),l5:1 Kb+ ladder, l5:Midiprep A digested with PstI & BamHI,l6:Midiprep A digested with SalI & BamHI, l7:Midiprep B digested with PstI & BamHI,l8:Midiprep B digested with SalI & BamHI, l9: 2 log ladder, l10: fragment of DelH - G3b


=> Don't trust the BB, because between 3 and 4 Kb there is no band. Backbone will be send for sequencing
=> Repeat entire cloning again?


Amplification of Backbone pSB6A1-lacZ-mRFP

As discussed, we are going to use another backbone. It is already in the parts registry: pSB6A1 + BBa_J04450 from Spring distribution 2012 (plate 1, well K1).

Transformation in E. coli TOP10

  • Chemical transformation of 3 µl of the plasmid
  • Incubation on ice, 15 min
  • Heat shock 42°C, 40 s
  • Incubation on ice again
  • Resuspended in 500 µl LB Amp for 30 min
  • Centrifuged (120, 5,000 rpm), removal of supernatant
  • Plated on LB Amp plate
  • Incubation ON at 37°C


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