Team:Heidelberg/Templates/Del week14 OP

From 2013.igem.org

(Difference between revisions)
(Created page with "==01-08-2013== ===Amplification from FS_22 to FS_13(s); 2.7 kb=== [[File:Heidelberg_20130802 log2 OPsslow OPlslowbesch.png|150px|thumb|Amplification of DelOP(FS22 to FS13short/l...")
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==30-07-2013==
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===Re-PCR of DelOP FS_22 to FS_13; 2.7 kb; [[DelO-P#19-07-2013|19-07-2013]])===
 +
 +
[[File:Heidelberg_20130730 3x 22-13long opecoR1 log2 Tyr 3 13 15besch.png|150px|thumb|Re-PCR OP, Restriction digest OP with EcoRI; run at 100 V, 0.8 % gel (TAE)]]
 +
 +
3x20µl
 +
:'''Reaction'''
 +
{| class="wikitable"
 +
|-
 +
! what !! µl 
 +
|-
 +
| Fragment FS_22 to FS_13_short ([[DelO-P#19-07-2013|19-07-2013]]) || 1
 +
|-
 +
| FS_22:  (1/10) || 2
 +
|-
 +
| FS_13_long:  (1/10) || 2
 +
|-
 +
| Phusion flash Master Mix || 10
 +
|-
 +
| dd H<sub>2</sub>O || 5
 +
|}
 +
 +
:'''Conditions'''
 +
{| class="wikitable"
 +
|-
 +
! colspan="3" | Biorad MyCycler*
 +
|-
 +
! Cycles !! temperature [°C] !! Time [s]
 +
|-
 +
| 1 || 98 || 10
 +
|-
 +
| rowspan="3"| 12 || 98 || 1
 +
|-
 +
| 65 ↓ 0.5  || 5
 +
|-
 +
| 72  || 1:00
 +
|-
 +
| rowspan="3"| 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 of fragment FS_22 to FS_13; 2.7 kb; [[DelO-P#25-07-2013|25-07-2013]]) with EcoRI-HF===
 +
[[File:Heidelberg_20130730 3x 22-13long opecoR1 log2 Tyr 3 13 15besch.png|150px|thumb|Re-PCR OP, Restriction digest OP with EcoRI; run at 100 V, 0.8 % gel (TAE)]]
 +
 +
Incubation at 37°C for
 +
{| class="wikitable"
 +
|-
 +
! what !! µl 
 +
|-
 +
| FS_22 to FS_13long([[DelO-P#25-07-2013|25-07-2013]]) || 17
 +
|-
 +
| EcoRI || 1
 +
|-
 +
| Buffer CutSmart || 2
 +
|-
 +
| Expected fragment lengths [bp] || 1883, 960
 +
|}
 +
 +
<div style="clear:both"></div>
 +
===Amplification from FS_22 to FS_13s; 2.7 kb===
 +
 +
[[File:Heidelberg_20130730 2log 3xre-pcr-FS22to13long FS22to13short2u-prim 2xFS22to13short4u-prim.png|300px|thumb|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]]
 +
[[File:Heidelberg_20130730 2log 3xre-pcr-FS22to13long FS22to13short2u-prim 2xFS22to13short4u-prim cut.png|300px|thumb|Gel after excision]]
 +
 +
2x20µl
 +
:'''Reaction'''
 +
{| class="wikitable"
 +
|-
 +
! what !! µl 
 +
|-
 +
| ''D.acidovorans'' || 1
 +
|-
 +
| FS_22:  (1/10) || 4
 +
|-
 +
| FS_13_short:  (1/10) || 4
 +
|-
 +
| Phusion flash Master Mix || 10
 +
|-
 +
| dd H<sub>2</sub>O || 1
 +
|}
 +
 +
20µl
 +
:'''Reaction'''
 +
{| class="wikitable"
 +
|-
 +
! what !! µl 
 +
|-
 +
| ''D.acidovorans'' || 1
 +
|-
 +
| FS_22:  (1/10) || 2
 +
|-
 +
| FS_13_short:  (1/10) || 2
 +
|-
 +
| Phusion flash Master Mix || 10
 +
|-
 +
| dd H<sub>2</sub>O || 5
 +
|}
 +
 +
:'''Conditions'''
 +
{| class="wikitable"
 +
|-
 +
! colspan="3" | Biorad MyCycler*
 +
|-
 +
! Cycles !! temperature [°C] !! Time [s]
 +
|-
 +
| 1 || 98 || 10
 +
|-
 +
| rowspan="3"| 12 || 98 || 1
 +
|-
 +
| 65 ↓ 0.5  || 5
 +
|-
 +
| 72  || 1:00
 +
|-
 +
| rowspan="3"| 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'''
 +
{| class="wikitable"
 +
|-
 +
! what !! µl 
 +
|-
 +
| ''D.acidovorans'' || 1
 +
|-
 +
| FS_22:  (1/10) || 2
 +
|-
 +
| FS_13_short/FS_13_long:  (1/10) || 2
 +
|-
 +
| Phusion flash Master Mix || 10
 +
|-
 +
|DMSO || 1/-
 +
|-
 +
| dd H<sub>2</sub>O || 4/5
 +
|}
 +
 +
:'''Conditions'''
 +
{| class="wikitable"
 +
|-
 +
! colspan="3" | Biorad C1000 Touch
 +
|-
 +
! Cycles !! temperature [°C] !! Time [s]
 +
|-
 +
| 1 || 98 || 10
 +
|-
 +
| rowspan="2"| 30 || 98 || 1
 +
|-
 +
| 72  || 1:00
 +
|-
 +
| 1 || 72 || 5 min
 +
|-
 +
| 1  || 12 || inf
 +
|}
 +
 +
:'''Conditions'''
 +
{| class="wikitable"
 +
|-
 +
! colspan="3" | Biorad MyCycler*
 +
|-
 +
! Cycles !! temperature [°C] !! Time [s]
 +
|-
 +
| 1 || 98 || 10
 +
|-
 +
| rowspan="3"| 30 || 98 || 1
 +
|-
 +
| 70  || 5
 +
|-
 +
| 72  || 1:00
 +
|-
 +
| 1 || 72 || 5 min
 +
|-
 +
| 1  || 12 || inf
 +
|}
 +
==01-08-2013==
==01-08-2013==

Revision as of 20:23, 30 September 2013

Contents

30-07-2013

Re-PCR of DelOP 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 of fragment FS_22 to FS_13; 2.7 kb; 25-07-2013) with EcoRI-HF

Re-PCR OP, Restriction digest OP with EcoRI; run at 100 V, 0.8 % gel (TAE)

Incubation at 37°C for

what µl
FS_22 to FS_13long(25-07-2013) 17
EcoRI 1
Buffer CutSmart 2
Expected fragment lengths [bp] 1883, 960

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 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 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 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

01-08-2013

Amplification from FS_22 to FS_13(s); 2.7 kb

Amplification of DelOP(FS22 to FS13short/long; 02.08); run at 100 V, 0.8 % gel (TAE)
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
dd H2O 5
Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
92 5
91 5
90 5
89 5
88 5
87 5
86 5
85 5
84 5
82 5
80 5
78 5
76 5
74 5
72 5
70 5
68 5
66 5
64 5
62 5
60 5
72 1:00
1 72 5 min
1 8 inf

Results:

  • Amplification of DelOP did not work with stepwise cooling to the intended annealing temperature of 60°C

07-08-2013

Amplification from FS_22 to FS_13; 2.7 kb

Amplifcation of DelOP and DelL (07.08); run at 100 V, 0.8 % gel (TAE)
Amplifcation of DelOP and DelL (07.08), cut
Reaction
what µl
D. acidovorans SPH-1 1
FS_22: (1/10) 2
FS_13: (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

Amplification from FS_22 to FS_13; 2.7 kb

Amplifcation of DelOP (55°C) (07.08); run at 100 V, 0.8 % gel (TAE)
Amplifcation of DelOP (55°C) (07.08)
Reaction
what µl
D. acidovorans SPH-1 1
FS_22: (1/10) 2
FS_13: (1/10) 2
Phusion flash Master Mix 10
dd H2O 5
Conditions
Biorad MyCycler*
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
55 5
72 1:00
1 72 5min
1 12 inf

Results:

  • Amplification of DelOP led to a band of the desired size as well as a smear and several different sideproducts
  • Band was cut out and DNA purified using QIAquick Gel Extraction Kit to be restriction digested for validation of the PCR product
  • Amplification will be repeated at lower temperature to obtain more product


Retrieved from "http://2013.igem.org/Team:Heidelberg/Templates/Del_week14_OP"