Team:Heidelberg/Templates/Del week14 FG

From 2013.igem.org

Contents

29-07-2013

Amplification from FS_21 to FS_24 ; (WRONG PRIMER!)

Amplifications of DelFG (29.07); run at 100 V, 0.8 % gel (TAE)
Amplifications of DelFG (29.07) cut
Reaction
what µl
D. acidovorans DSM-39 1
FS_21: (1/10) 2
FS_24: (1/10) 2
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions
Biorad MyCycler*
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
55 5
72 2:30
1 72 8 min
1 12 inf

Results:

  • PCR product occured, though wrong primers were used, unspecific binding of primers in the genome of D. Acidovorans is the putative reason for this event

Amplification from FS_21 to FS_24; (WRONG PRIMER!)

Amplification of DelFG (FS21 to FS26; 29.07); run at 100 V, 0.8 % gel (TAE)
Amplification of DelFG (FS21 to FS26; 29.07) cut
Reaction
what µl
D. acidovorans DSM-39 1
FS_21: (1/10) 2
FS_24: (1/10) 2
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
58 5
72 2:30
1 72 8 min
1 12 inf

Results:

  • PCR product occured, though wrong primers were used, unspecific binding of primers in the genome of D. Acidovorans is the putative reason for this event

Amplification from FS_21 to FS_24; (WRONG PRIMER!)

Amplification of DelFG (FS21 to FS26; 29.07); run at 100 V, 0.8 % gel (TAE)
Amplification of DelFG (FS21 to FS26; 29.07) cut
Reaction
what µl
D. acidovorans DSM-39 1
FS_21: (1/10) 2
FS_24: (1/10) 2
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
35 98 1
60 5
72 2:30
1 72 8 min
1 12 inf

Results:

  • PCR product occured, though wrong primers were used, unspecific binding of primers in the genome of D. Acidovorans is the putative reason for this event

Amplification from FS_06/FS_20/FS_21 to FS_26 ; 5.5 kb

Amplifications of DelFG (29.07); run at 100 V, 0.8 % gel (TAE)
Amplifications of DelFG (29.07) cut


3x20µl

Reaction
what µl
D. acidovorans DSM-39 1
FS_06 or FS_20 or FS_21: (1/10) 2
FS_26: (1/10) 2
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
64 5
72 2:15
1 72 10 min
1 10 inf

Results:

  • PCR product occured, though wrong primers were used, unspecific binding of primers in the genome of D. Acidovorans is the putative reason for this event
  • Furthermore, amplification with FS_21 to FS_26 led to the intended product and satisfying specifity
  • Amplification will be repeated at the same annealing temperature to obtain the amount of PCR product required for Gibson Assembly
  • Amplfication will be repeated at lower annealing temperature to increase the yield
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit

30-07-2013

Amplification from FS_21 to FS_26 ; 5.5 kb

Amplification of DelFG (FS21 to FS26; 30.07) at 60°C and 64°C; run at 100 V, 0.8 % gel (TAE)
Amplification of DelFG (FS21 to FS26; 30.07) cut

3x20µl with conditions I, 2x20µl with conditions II

Reaction
what µl
D. acidovorans DSM-39 1
FS_21: (1/10) 2
FS_26: (1/10) 2
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions I
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
64 5
72 2:15
1 72 10 min
1 10 inf
Conditions II
Biometra TProfessional Basic
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
60 5
72 2:15
1 72 10 min
1 10 inf

Results:

  • Amplification of DelFG was successful
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit
  • restriction digest with XmaI will be conducted to validate the PCR product

31-07-2013

Restriction digest of fragment FS_21 to FS_26 (5.5 kb; 29-07-2013) with XmaI

Restriction digest of FS21 to FS26 (29.7) with XmaI; run at 100 V, 0.8 % gel (TAE)

Incubation at 37°C for about 3 hours

what µl
FS_21 to FS_26 (29-07-2013) 17
XmaI 1
Buffer CutSmart 2
Expected fragment lengths [bp] 4268, 1202

Results:

  • Restriction digest of DelFG with XmaI did not lead to the expected result
  • digest will be carried out with a different enzyme, as the used one was outdated and digest might therefore not be very reliable

02-08-2013

Restriction digest of fragment FS_21 to FS_26 (5.5 kb; 30-07-2013) with ClaI

Restriction digest of FS21 to FS26 (30.7) with ClaI; run at 100 V, 0.8 % gel (TAE)

Incubation at 37°C for about 3 hours

what µl
FS_21 to FS_26 (30-07-2013) ~15
ClaI 1
Buffer CutSmart 2
Expected fragment lengths [bp] 2743, 1519, 1208

Results:

  • Restriction digest of DelFG did not display any product
  • digest will be repeated with higher amount of DNA after new amplification from the genome of D. Acidovorans

07-08-2013

Amplification from FS_21 to FS_26 ; 5.5 kb

Amplifcation of DelAF(I), DelFG, DelG (07.08); run at 100 V, 0.8 % gel (TAE)
Amplifcation of DelAF(I), DelFG, DelG (07.08), cut
Reaction
what µl
D. acidovorans SPH-1 1
FS_21: (1/10) 2
FS_26: (1/10) 2
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions
Biorad MyCycler
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
60 5
72 2:15
1 72 10 min
1 10 inf

Results:

  • Amplification of DelFG was successful, gel displays highly specific product of convincing yield
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit
  • restriction digest with ClaI will be conducted to validate PCR product
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