Team:Heidelberg/Templates/Del week14 OP

From 2013.igem.org

Contents

30-07-2013

Re-PCR of DelOP FS_22 to FS_13; 2.7 kb; 19-07-2013

Re-PCR OP, Restriction digest OP with EcoRI; run at 100 V, 0.8 % gel (TAE)

3x20µl

Reaction
what µl
Fragment FS_22 to FS_13_short (19-07-2013) 1
FS_22: (1/10) 2
FS_13_long: (1/10) 2
Phusion flash Master Mix 10
dd H2O 5
Conditions
Biorad MyCycler*
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
65 ↓ 0.5 5
72 1:00
18 98 1
63 5
72 1:00
1 72 5min
1 12 inf

Results:

  • Amplification of DelOP did not work
  • PCR will be repeated with different primer concentrations to estimate whether primer dimes might be the reason for insufficient amplification

Restriction digest of fragment FS_22 to FS_13; 2.7 kb; 25-07-2013 with EcoRI-HF

Re-PCR OP, Restriction digest OP with EcoRI; run at 100 V, 0.8 % gel (TAE)

Incubation at 37°C for

what µl
FS_22 to FS_13long(25-07-2013) 17
EcoRI 1
Buffer CutSmart 2
Expected fragment lengths [bp] 1883, 960

Amplification from FS_22 to FS_13s; 2.7 kb

Amplification of Del O-P from FS_22 to FS_13long/FS_13short under different conditions run at 100 V 0.8% agarose gel (TAE
Gel after excision

2x20µl

Reaction
what µl
D.acidovorans 1
FS_22: (1/10) 4
FS_13_short: (1/10) 4
Phusion flash Master Mix 10
dd H2O 1

20µl

Reaction
what µl
D.acidovorans 1
FS_22: (1/10) 2
FS_13_short: (1/10) 2
Phusion flash Master Mix 10
dd H2O 5
Conditions
Biorad MyCycler*
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
65 ↓ 0.5 5
72 1:00
18 98 1
63 5
72 1:00
1 72 5min
1 12 inf

Results:

  • Amplification of DelOP resulted in a small band atthe desired lenght, but also a smear and several unexpected bands
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit
  • amplicon will be used for restriction digest to validate the construct

Amplification from FS_22 to FS_13(s/l); 2.7 kb

Reaction
what µl
D.acidovorans 1
FS_22: (1/10) 2
FS_13_short/FS_13_long: (1/10) 2
Phusion flash Master Mix 10
DMSO 1/-
dd H2O 4/5
Conditions
Biorad C1000 Touch
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
72 1:00
1 72 5 min
1 12 inf
Conditions
Biorad MyCycler*
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
70 5
72 1:00
1 72 5 min
1 12 inf

01-08-2013

Amplification from FS_22 to FS_13(s); 2.7 kb

Amplification of DelOP(FS22 to FS13short/long; 02.08); run at 100 V, 0.8 % gel (TAE)
Reaction
what µl
D.acidovorans DSM-39 1
FS_22: (1/10) 2
FS_13_short/FS_13_long: (1/10) 2
Phusion flash Master Mix 10
dd H2O 5
Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
92 5
91 5
90 5
89 5
88 5
87 5
86 5
85 5
84 5
82 5
80 5
78 5
76 5
74 5
72 5
70 5
68 5
66 5
64 5
62 5
60 5
72 1:00
1 72 5 min
1 8 inf

Results:

  • Amplification of DelOP did not work with stepwise cooling to the intended annealing temperature of 60°C

07-08-2013

Amplification from FS_22 to FS_13; 2.7 kb

Amplifcation of DelOP and DelL (07.08); run at 100 V, 0.8 % gel (TAE)
Amplifcation of DelOP and DelL (07.08), cut
Reaction
what µl
D. acidovorans SPH-1 1
FS_22: (1/10) 2
FS_13: (1/10) 2
Phusion flash Master Mix 10
dd H2O 5
Conditions
Biorad MyCycler*
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
65 ↓ 0.5 5
72 1:00
18 98 1
63 5
72 1:00
1 72 5min
1 12 inf

Amplification from FS_22 to FS_13; 2.7 kb

Amplifcation of DelOP (55°C) (07.08); run at 100 V, 0.8 % gel (TAE)
Amplifcation of DelOP (55°C) (07.08)
Reaction
what µl
D. acidovorans SPH-1 1
FS_22: (1/10) 2
FS_13: (1/10) 2
Phusion flash Master Mix 10
dd H2O 5
Conditions
Biorad MyCycler*
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
55 5
72 1:00
1 72 5min
1 12 inf

Results:

  • Amplification of DelOP led to a band of the desired size as well as a smear and several different sideproducts
  • Band was cut out and DNA purified using QIAquick Gel Extraction Kit to be restriction digested for validation of the PCR product
  • Amplification will be repeated at lower temperature to obtain more product


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