Team:Heidelberg/Templates/Del week15 OP

From 2013.igem.org

Contents

08-08-2013

Amplification from FS_22 to FS_13, 2.7 kb

Amplifcation of DelAE and DelEF and DelOP; run at 100 V, 0.8 % gel (TAE)(07.08)
Amplifcation of DelAE and DelEF and DelOP; run at 100 V, 0.8 % gel (TAE)(07.08)


Reaktion
what µl
D. acidovorans SPH-1 (colony-pcr) 1
FS_22: (1/10) 4
FS_13long: (1/10) 4
Phusion flash Master Mix 10
Conditions
Biorad MyCycler*
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
50 5
72 1:00
1 72 5 min
1 10 inf

Results:

  • Amplification of DelOP did not work
  • PCR will be repeated at a higher annealing temperature of 65°C to increase primer binding

Restriction digest of fragment FS_22 to FS_13; 2.7 kb; 07-08-2013) with EcoRI-HF

Restriction digest of DelG (SR01-FS23), DelG (FS21-FS26) and DelOP; run at 100 V, 0.8 % gel (TAE)(07.08)

Incubation at Room temperature for about 4h and at 37°C for 1 hours

what µl
SR_22 to FS_13 (07-08-2013) 20
EcoRI-HF 1
Buffer CutSmart 3
dd H2O 6
Expected fragment lengths [bp] 1883, 960

Results:

  • Restriction digest did not work, amplicon was not cut and total amount of DNA was probably too low
  • Restriction digest will be repeated as soon as PCR of DelOP works reliable and reproducable with higher amounts of DNA

Concentration measurement OP (07-08-2013)

Fragment Primer Date PCR Concentration
DelOP FS22-FS13 07-08-2013 6.1 ng/µl

09-08-2013

Amplification I from FS_22 to FS_13, 2.7 kb

Amplifcation of DelOP (I), DelFG and DelL ; run at 100 V, 0.8 % gel (TAE)(09.08)
Amplifcation of DelOP (I), DelFG and DelL ; run at 100 V, 0.8 % gel (TAE)(09.08)



Reaktion
what µl
D. acidovorans SPH-1 (colony-pcr) 1
FS_22: (1/10) 4
FS_13long: (1/10) 4
Phusion flash Master Mix 10
Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
65 5
72 1:00
1 72 5 min
1 10 inf

Results:

  • Amplification of DelOP did not work
  • PCR will be repeated at a lower annealing temperature of 55°C in case primers were not able to bind at high temperatures

Amplification II from FS_22 to FS_13; 2.7 kb

Amplifcation of DelOP (II), from glycerol stocks at 55°C const. ; run at 100 V, 0.8 % gel (TAE)(09.08)



wrong Primers were used

Reaktion
what µl
D. acidovorans SPH-1 (colony-pcr) 1
FS_22: (1/10) 4
FS_13long: (1/10) 4
Phusion flash Master Mix 10
Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
55 5
72 1:00
1 72 5 min
1 10 inf

Results:

  • Amplification of DelOP did not work as the wrong primers were used
  • Amplification will be repeated with different amounts of DMSO

Amplification III from FS_22 to FS_13; 2.7 kb

Amplifcation of DelOP (III), from agar plate at 55°C const. ; run at 100 V, 0.8 % gel (TAE)(09.08)

3 samples, one with, two without DMSO (one with much template, one with less template)

Reaktion
what µl
D. acidovorans SPH-1 colony 1
FS_22: (1/10) 4
FS_13long: (1/10) 4
Phusion flash Master Mix 10
DMSO 0/1
Conditions
Biometra TProfessional Basic
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
55 5
72 1:00
1 72 5 min
1 10 inf

Results:

  • Amplification of DelOP did not work, neither with lower amount of template, nor with higher, furthermore the DMSO concentration did not influence the PCR for the given annealing temperature
  • PCR will be repeated with recently ordered new Primers which bind outside the intended template in the Del-Cluster of D. acidovorans SPH-1 in order to obtain a specific template suited for a nested PCR of DelOP with Primers FS_22 and FS_14l

10-08-2013

Amplification I, nested PCR different Primers; 2.7 kb

Amplifcation I of DelOP ; run at 135 V, 0.8 % gel (TAE)(10.08)
Reaction
Reagent DelOP
Template D.acidovorans SPH-1 colony
Primer fw 4 µl FS_32 4 µl FS_32 4 µl FS_32 4 µl FS_32 4 µl FS_32
Primer rev 4 µl FS_13l 4 µl FS_27 4 µl FS_28 4 µl FS_29 4 µl FS_30
Phusion Ready Mix 10 µl
dd H2O 1 µl
Conditions
Biometra TProfessional Basic
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
55 5
72 1:00
1 72 5 min
1 10 inf

Results:

  • Amplification of DelOP did not work
  • PCR will be repeated as touchdown PCR with annealing starting at 68°C and different primer combinations to the template needed for the nested PCR of DelOP with primers FS_22 to FS_13l

Amplification II, nested PCR with different primers; 2.7 kb

Amplifcation II of DelOP ; run at 135 V, 0.8 % gel (TAE)(10.08)
Amplifcation II of DelOP after cutting ; run at 135 V, 0.8 % gel (TAE)(10.08)
Reaction
Reagent DelOP
Template D.acidovorans SPH-1 colony
Primer fw 4 µl FS_22 4 µl FS_22 4 µl FS_33 4 µl FS_33 4 µl FS_31 4 µl FS_31 4 µl FS_32 4 µl FS_32 4 µl FS_22 4 µl FS_22
Primer rev 4 µl FS_13s 4 µl FS_27 4 µl FS_13s 4 µl FS_27 4 µl FS_29 4 µl FS_30 4 µl FS_29 4 µl FS_30 4 µl FS_28 4 µl FS_29
DMSO 1 µl
Phusion Ready Mix 10 µl
dd H2O 1 µl
Conditions
Biometra TProfessional Basic
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 1:00
18 98 1
65 5
72 1:00
1 72 10min
1 12 inf

Results:

  • Amplification of DelOP worked with the Primer combinations FS22-FS13short, FS_22-FS_27, FS_33-FS_13short, FS_33-FS_27, FS_32-FS_30 and FS_22-FS28
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit
  • all the obtained PCR products will be used as specific templates for a nested PCR using primers FS_22-FS_13long

Amplification III from FS_22 to FS_13; 2.7 kb

Amplifcation of DelOP and DelAF ; run at 135 V, 0.8 % gel (TAE)(10.08)
Amplifcation of DelOP and DelAF after cutting ; run at 135 V, 0.8 % gel (TAE)(10.08)
Reaction
Reagent DelOP
Template D.acidovorans SPH-1 colony
Primer fw 4 µl FS_22
Primer rev 4 µl FS_13l
DMSO 1 µl
Phusion Ready Mix 10 µl
dd H2O 1 µl
Conditions
Biometra TProfessional Basic
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 1:00
18 98 1
65 5
72 1:00
1 72 10min
1 10 inf

Results:

  • Amplification of DelOP did not work
  • hope and wait for the nested PCR

11-08-2013

Nested PCR of DelOP-Fragments from 10-08-2013

quantification of DelOP PCR-products from 10.08 after gel extraction to determine templates for followed nested PCR; run at 135 V, 0.8 % gel (TAE)(11.08)
Nested PCR of DelOP using specific PCR-products from 10.08; run at 135 V, 0.8 % gel (TAE)(11.08)
Reaction
Reagent DelOP
Template FS22-13s FS31-30 FS33-27 FS22-28 FS22-13s FS31-30 FS33-29
Primer fw 4.5 µl FS_22
Primer rev 4.5 µl FS_13l
DMSO 1 µl
Phusion Ready Mix 10 µl


Conditions


Biometra TProfessional Basic
Cycles temperature [°C] Template FS22-13s, FS31-30, FS33-27, FS22-28 Time Cycles temperature [°C] Template FS22-13s, FS31-30, FS33-29, Time
1 98 10 s 1 98 10 s
30 98 1 s 12 98 1 s
68 ↓ 0.5 5 s
58 5 s 72 1:00 min
18 98 1 s
72 1:00 min 66 5 s
72 1:00 min
1 72 5 min 1 72 5 min
1 10 inf 1 10 inf

Results:

  • Nested PCR of DelOP did not work, though concentrations of the used template were fine as checked previously
  • PCR will be with an annealing temperature of 55°C with the template obtained from the primer combination of FS_22-FS_13short as nested PCR
Retrieved from "http://2013.igem.org/Team:Heidelberg/Templates/Del_week15_OP"