Team:Heidelberg/Templates/Del week16 OP

From 2013.igem.org

Contents

12-08-2013

Amplification I from FS_22 to FS_13; 2.7 kb

Reaction
Reagent DelOP
Template D.acidovorans SPH-1 colony 1µl FS_22-FS_13_short 10-08
Primer fw 2 µl FS_22
Primer rev 2 µl FS_13
Phusion Ready Mix 10 µl
dd H2O 5 µl
Conditions
Biorad MyCycler*
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
55 5
72 1:00
1 72 5min
1 10 inf

Results:

  • Amplification of DelOP did not work
  • PCR will be repeated at very high annealing temperatures to ensure that primers with very complex secondary structure such as the Gibson-Primer FS_13long are able to bind

Amplification II and III from FS_22 to FS_13; 2.7 kb

Amplification of DelOP III .; run at 100 V, 0.8 % gel (TAE)(12.08)


Amplification of DelOP 72°C const.; run at 135 V, 0.8 % gel (TAE)(11.08)
Amplification of DelOP 72°C const. after cutting; run at 135 V, 0.8 % gel (TAE)(11.08)
Reaction
Reagent DelOP
Template 1µl FS_22-FS_13_short 10.08
Primer fw 4 µl FS_22
Primer rev 4 µl FS_13
Phusion Ready Mix 10 µl
DMSO 1 µl
Conditions
Biorad MyCycler
Cycles temperature [°C] DelOP II Time Cycles temperature [°C] DelOP III Time
1 98 10 s 1 98 10 s
30 98 1 s 12 98 1 s
74.0 - 70.0 (ΔT = 2.0) ↓ 0.5 5 s
72 1:00 min 72 3:15 min
18 98 1 s
66 5 s
68 / 70 / 72 1:00 min
1 72 10 min 1 72 10 min
1 10 inf 1 10 inf

Results:

  • Amplification of DelOP was successful, 2-step PCR with FS_22-FS_13s as template led to expected bands as well as a slight smear and an unintended product
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit
  • PCR will be repeated to obtain more sample

Re-PCR from FS_22 to FS_13; 2.7 kb; 10-08-2013)

Amplification of DelAF using gradient PCR, Amplification of DelOP 72°C 2-step; run at 135 V, 0.8 % gel (TAE)(11.08)
Amplification of DelAF using gradient PCR, Amplification of DelOP 72°C 2-step after cutting; run at 135 V, 0.8 % gel (TAE)(11.08)


3x 20µl

Reaction
Reagent DelOP
Template 1µl FS_22-FS_13_short 10-08-2013
Primer fw 4 µl FS_22
Primer rev 4 µl FS_13
Phusion Ready Mix 10 µl
DMSO 1 µl
Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
72 1:05
1 72 5 min
1 10 inf

Results:

  • Amplification of DelOP was successfull
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit
  • gradient PCR with annealing at 74°C, 72°C and 70°C will be carried out to increase yield

Amplification V from FS_22 to FS_13; 2.7 kb

Amplification of DelOP V (12-08-13); run at 100 V, 0.8 % gel (TAE)
Amplification of DelOP V (12-08-13) cut
Reaction
Reagent DelOP
Template D.acidovorans SPH-1 colony
Primer fw 4.5 µl FS_22
Primer rev 4.5 µl FS_13
Phusion Ready Mix 10 µl
DMSO 1 µl
Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
75.0 - 72.0 (ΔT = 0.5) 1:05
1 72 5 min
1 10 inf

Results:

  • Amplification of DelOP was successfull
  • The gradient PCR shows that amplification worked best with an annealing temperature of 72.3°C, interestingly this annealing temperature was used for the remaining mastermix of the gradient PCR which included more DMSO than the other samples as mixing of DMSO often is not perfect and therefore the very last sample is of higher DMSO concentration
  • Therefore PCR will be repeated with an annealing temperature of 72.3°C and 10% DMSO

13-08-2013

Restriction digest of fragment FS_22 to FS_13; 2.7 kb; 12-08-2013) with EcoRI-HF

Incubation at 37°C for 2 hours

what µl
FS_22 to FS_13 (12-08-2013) 20
EcoRI-HF 1
CutSmart Buffer 2.5
dd H2O 1.5
Expected fragment lengths [bp] 1883, 960


Results:

  • restriction digest shows the expected bands, therefore validation if PCR amplicon is sufficient for Gibson Assembly

Amplification from FS_22 to FS_13; 2.7 kb

Amplification of DelAF (FS_02-FS_05); run at 100 V, 0.8 % gel (TAE)(13.08)
Amplification of DelAF (FS_02-FS_05), cut; run at 100 V, 0.8 % gel (TAE)(13.08)
Reaction
Reagent DelOP
Template D.acidovorans SPH-1 colony
Primer fw 4 µl FS_22
Primer rev 4 µl FS_13
Phusion Ready Mix 10 µl
DMSO 1/2 µl
dd H2O 1/- µl
Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
72 1:05
1 72 5 min
1 10 inf

Results:

  • Amplification of DelOP was successfull with 10% DMSO leading to one specific band of intended size
  • PCR will be repeated with 10% DMSO to obtain the amount of product which is necessary for Gibson Assembly and test restriction digest

14-08-2013

Concentration measurement DelOP

Fragment Primer Date PCR Concentration
DelOP FS22-FS13 12-08-2013 11 ng/µl

17-08-2013

Amplification from FS_22 to FS_13; 2.7 kb

6x20 µl

Reaction
Reagent DelOP
Template D.acidovorans SPH-1 colony
Primer fw 2.5 µl FS_22
Primer rev 2.5 µl FS_13
Phusion Ready Mix 10 µl
DMSO 2 µl
dd H2O 3 µl


Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
72.3 5
72 1:00
1 72 5 min
1 10 inf

18-08-2013

Amplification from FS_22 to FS_13; 2.7 kb

Restriction digest of pSB4K5 (17-08) with DpnI (17-08) and amplification of DelOP (18-08); run at 100 V, 0.8 % gel (TAE)
Restriction digest of pSB4K5 (17-08) with DpnI (17-08) and amplification of DelOP, cut (18-08); run at 100 V, 0.8 % gel (TAE)

6x20 µl

Reaction
Reagent DelOP
Template D.acidovorans SPH-1 colony
Primer fw 2.5 µl FS_22
Primer rev 2.5 µl FS_13
Phusion Ready Mix 10 µl
DMSO 2 µl
dd H2O 3 µl


Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
72.3 5
72 1:00
1 72 5 min
1 10 inf

Results:

  • Amplification of DelOP was successful
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit
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