Team:Heidelberg/Templates/Del week13 OP

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Contents

25-07-2013

Re-PCR I from FS_22 to FS_13; 2.7 kb; 19-07-2013

Re-PCR I of OP (FS_22 to FS_13_long); 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) 4
FS_13_long: (1/10) 4
Phusion flash Master Mix 10
dd H2O 1
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 led to a smear and several unexpected bands
  • Re-PCR will be repeated

Re-PCR I from FS_22 to FS_13; 2.7 kb; 19-07-2013

4 x Amplification of DelFG (FS20 to FS07; 26.07), 4 x Amplification of DelOP (FS_22 to FS13; 25.07); run at 100 V, 0.8 % gel (TAE)
4 x Amplification of DelFG (FS20 to FS07; 26.07), 4 x Amplification of DelOP (FS_22 to FS13; 25.07) after cutting; 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:

  • a smear as well as a band of the expeceted size occured
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit
  • DNA will be used for restriction digests to validate amplicon

26-07-2013

Restriction digest of fragment FS_22 to FS_13s; 2.7 kb; 21-07-2013 with EcoRI-HF

Restriction digest of DelAE (FS_02 to FS_03 ;08.07), DelAF (FS_02 to FS_05; 04.07) and DelOP (FS_22 to FS_13short ;21.07) with EcoRI-HF and digest of DelG (FS_08 to FS_23 cut1 ;23.07) with BglII; run at 100 V, 0.8 % gel (TAE)

Incubation at 37°C for 1h 45min

what µl
FS_22 to FS_13short (21-07-2013) 20
EcorRI-HF 0.8
CutSmart 2.5
dd H2O 2.5
Expected fragment lengths [bp] 1883, 960

Results:

  • Restriction digest did not work, the amplified fragment was either not cut or the fragments are not visible due to a very small amount of DNA
  • Re-PCR will be repeated to obtain the higher amounts of DNA required for the restriction digest

30-07-2013

Re-PCR from 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 from FS_22 to FS_13; 2.7 kb; 25-07-2013 with EcoRI-HF

Incubation at 37°C for

what µl
FS_22 to FS_13long(25-07-2013) 17
EcoRI 1
Buffer CutSmart 2

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 DSM-39 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 DSM-39 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 DSM-39 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
Retrieved from "http://2013.igem.org/Team:Heidelberg/Templates/Del_week13_OP"