Team:Heidelberg/Templates/Del week13 G

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Contents

22-07-2013

Re-PCR from FS_10 to FS_23; 3.3 kb; 13-07-2013

Re-PCR of DelG (10-23), Amplification of DelG (8-23) (22.07); run at 100 V, 0.8 % gel (TAE)
Re-PCR of DelG (10-23), Amplification of DelG (8-23) (22.07) cut; run at 100 V, 0.8 % gel (TAE)
Reaction
what µl
Fragment FS_10 to FS_11_short (13-07-2013) 1
FS_10: (1/10) 2
FS_23: (1/10) 2
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions

--> First cycles with wrong program

Biorad C1000 Touch Block A
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 1:10 min
18 98 1
66 5
72 1:10 min
1 72 5 min
1 12 inf

Results:

  • Re-Amplification of DelG lead to the expected fragment but also to a smear, therefore fragment will not be used for Gibson Assembly but restriction digests to validate the product.

Amplification from FS_08 to FS_23; 6.5 kb

Reaction
what µl
D. acidovorans DSM-39 1
FS_08: (1/10) 2
FS_23: (1/10) 2
Phusion flash Master Mix 10
DMSO 1/-
dd H2O 4/-
Conditions
Biorad MyCycler*
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
66 ↓ 0.5 5
72 3:20
18 98 1
64 5
72 3:20
1 72 10min
1 12 inf

Results:

  • Amplification of DelG worked, leading to the expected product as well one other unexpected band.
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit

Amplification from FS_08 to FS_11s; 6.5 kb

Reaction
what µl
D. acidovorans DSM-39 1
FS_08: (1/10) 2
FS_11_short: (1/10) 2
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions
Biometra TProfessional Basic
Cycles temperature [°C] Time [s]
1 98 10
14 98 1
62 ↓ 0.5 5
72 3:20
16 98 1
60 5
72 3:20
1 72 10min
1 12 inf

23-07-2013

Amplification from FS_08 to FS_23; 6.5 kb

Amplification of DelG (FS_08-FS_23); run at 100 V, 0.8 % gel (TAE)
Amplification of DelG (FS_08-FS_23)after cutting; run at 100 V, 0.8 % gel (TAE)
Measurement of concentration of DelG, DelFG and DelOP (2µl of each); run at 100 V, 0.8 % gel (TAE)


4x 20µl

Reaction
what µl
D. acidovorans DSM-39 1
FS_08: (1/10) 2
FS_23: (1/10) 2
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions
Biometra TProfessional Basic
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
66 ↓ 0.5 5
72 3:20
18 98 1
64 5
72 3:20
1 72 10min
1 12 inf

Results:

  • Amplification of DelG was successful
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit
  • Gel to proof quality of gel extraction was run

26-07-2013

Restriction digest of Fragment FS_08 to FS_23; 6.5 kb; 23-07-2013 with BglII

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 1hour 45min

what µl
FS_08 to FS_23 cut1 (23-07-2013) 15
BglII 0.5
Buffer 3.1 2
dd H2O 2

Results:

  • Restriction digest of DelG (FS_08 to FS_23) was successful though one unexpected band (presumably carry over) appeared
  • Construct will be further validated by sequencing before potential Gibson Assembly
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