http://2013.igem.org/wiki/index.php?title=Team:Heidelberg/Templates/Del_week16_pSB4K5&feed=atom&action=history
Team:Heidelberg/Templates/Del week16 pSB4K5 - Revision history
2024-03-28T10:48:03Z
Revision history for this page on the wiki
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http://2013.igem.org/wiki/index.php?title=Team:Heidelberg/Templates/Del_week16_pSB4K5&diff=259801&oldid=prev
Charly at 13:55, 1 October 2013
2013-10-01T13:55:08Z
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<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 13:55, 1 October 2013</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==15-08-2013==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==15-08-2013==</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Amplification from FS_01 to FS_16; 4.2 kb===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Amplification from FS_01 to FS_16; 4.2 kb===</div></td></tr>
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Charly
http://2013.igem.org/wiki/index.php?title=Team:Heidelberg/Templates/Del_week16_pSB4K5&diff=251637&oldid=prev
Charly: Created page with "==15-08-2013== ===Amplification from FS_01 to FS_16; 4.2 kb=== [[File:Heidelberg_20130814 2log 4xDelFGstandard 2log 4xpSB4K5standard.png|150px|thumb| Amplification of DelFG and ..."
2013-09-30T01:25:27Z
<p>Created page with "==15-08-2013== ===Amplification from FS_01 to FS_16; 4.2 kb=== [[File:Heidelberg_20130814 2log 4xDelFGstandard 2log 4xpSB4K5standard.png|150px|thumb| Amplification of DelFG and ..."</p>
<p><b>New page</b></p><div>==15-08-2013==<br />
===Amplification from FS_01 to FS_16; 4.2 kb===<br />
<br />
[[File:Heidelberg_20130814 2log 4xDelFGstandard 2log 4xpSB4K5standard.png|150px|thumb| Amplification of DelFG and Backbone pSB4K5; run at 100 V, 0.8 % gel (TAE)(14.08)]]<br />
[[File:Heidelberg_20130814 2log 4xDelFGstandard 2log 4xpSB4K5standard cut.png|150px|thumb| Amplification of DelFG and Backbone pSB4K5; run at 100 V, 0.8 % gel (TAE)(14.08)]]<br />
<br />
:'''Reaction''' <br />
(2x50µl)<br />
{| class="wikitable"<br />
|-<br />
! what !! µl <br />
|-<br />
| Template pSB4K5 || 1<br />
|-<br />
| FS_01: (1/10) || 2.5<br />
|-<br />
| FS_16: (1/10) || 2.5<br />
|-<br />
| Phusion Master Mix || 25<br />
|-<br />
| DMSO || 2.5<br />
|-<br />
| dd H<sub>2</sub>O || 16.5<br />
|}<br />
<br />
<br />
:'''Conditions'''<br />
{| class="wikitable"<br />
|-<br />
! colspan="3" | Biorad T100<br />
|-<br />
! Cycles-PCR !! temperature [°C] !! Time [s]<br />
|-<br />
| 1 || 98 || 5<br />
|-<br />
| rowspan="3"| 12 || 98 || 1<br />
|-<br />
| 62 || 5<br />
|-<br />
| 72 || 1:30 min<br />
|-<br />
| 1 ||72 || 5 min<br />
|-<br />
| 1 || 8 || inf<br />
|}<br />
<br />
'''Results:'''<br />
* Amplification worked very well, we had bright bands on the right height.<br />
* Bands were cut out and DNA purified using QIAquick Gel Extration Kit.<br />
<br />
===Restriction digest of pSB4K5 (FS_01 to FS_16; 4.2 kb; [[PSB4K5#15-08-2013|15-08-2013]]) with DpnI===<br />
<br />
Incubation at 37°C for about 6 hours<br />
{| class="wikitable"<br />
|-<br />
! what !! µl <br />
|-<br />
| FS_16 to FS_01 ([[PSB4K5#15-08-2013|15-08-2013]]) || 30<br />
|-<br />
| DpnI || 2<br />
|-<br />
| CutSmart Buffer || 4<br />
|-<br />
|dd H<sub>2</sub>O || 4<br />
|}<br />
<br />
'''Results:'''<br />
* Bands were cut out and DNA purified using QIAquick Gel Extration Kit.<br />
<br />
==16-08-2013==<br />
===Concentration measurement===<br />
The concentration of the gel purified, and DpnI digested fragment was measured using a NanoDrop Instrument.<br />
{| class="wikitable"<br />
|-<br />
! Fragment !! Primer !! Date PCR !! Concentration<br />
|-<br />
| pSB4K5 DpnI digested || FS01-FS16 || [[PSB4K5#15-08-2013|15-08-2013]] || 30 ng/µl<br />
|}<br />
<br />
===Amplification from FS_01 to FS_16; 4.2 kb===<br />
<br />
[[File:Heidelberg_20130817 1kbplus TetII 2log 7xpSB4K5besch.png|150px|thumb| Amplification of pSB4K5 (16-08); run at 100 V, 0.8 % gel (TAE)]]<br />
[[File:Heidelberg_20130817 1kbplus TetII 2log 7xpSB4K5 cutbesch.png|150px|thumb| Amplification of pSB4K5; cut (16-08); run at 100 V, 0.8 % gel (TAE)]]<br />
<br />
:'''Reaction''' <br />
(3x50µl)<br />
{| class="wikitable"<br />
|-<br />
! what !! µl <br />
|-<br />
| Template pSB4K5 || 1<br />
|-<br />
| FS_01: (1/10) || 2.5<br />
|-<br />
| FS_16: (1/10) || 2.5<br />
|-<br />
| Phusion Master Mix || 25<br />
|-<br />
| DMSO || 2.5<br />
|-<br />
| dd H<sub>2</sub>O || 16.5<br />
|}<br />
<br />
<br />
:'''Conditions'''<br />
{| class="wikitable"<br />
|-<br />
! colspan="3" | Biorad T100<br />
|-<br />
! Cycles-PCR !! temperature [°C] !! Time [s]<br />
|-<br />
| 1 || 98 || 5<br />
|-<br />
| rowspan="3"| 12 || 98 || 1<br />
|-<br />
| 62 || 5<br />
|-<br />
| 72 || 1:30 min<br />
|-<br />
| 1 ||72 || 5 min<br />
|-<br />
| 1 || 8 || inf<br />
|}<br />
<br />
<br />
'''Results:'''<br />
* Amplification worked very well, we had bright bands on the right height.<br />
* Bands were cut out and DNA purified using QIAquick Gel Extration Kit.<br />
<br />
==17-08-2013==<br />
<br />
<br />
===Amplification from FS_01 to FS_16; 4.2 kb===<br />
<br />
:'''Reaction''' <br />
(3x50µl)<br />
{| class="wikitable"<br />
|-<br />
! what !! µl <br />
|-<br />
| Template pSB4K5 || 1<br />
|-<br />
| FS_01: (1/10) || 2.5<br />
|-<br />
| FS_16: (1/10) || 2.5<br />
|-<br />
| Phusion Master Mix || 25<br />
|-<br />
| DMSO || 2.5<br />
|-<br />
| dd H<sub>2</sub>O || 16.5<br />
|}<br />
<br />
<br />
:'''Conditions'''<br />
{| class="wikitable"<br />
|-<br />
! colspan="3" | Biorad MyCycler<br />
|-<br />
! Cycles-PCR !! temperature [°C] !! Time [s]<br />
|-<br />
| 1 || 98 || 5<br />
|-<br />
| rowspan="3"| 12 || 98 || 1<br />
|-<br />
| 62 || 5<br />
|-<br />
| 72 || 1:30 min<br />
|-<br />
| 1 ||72 || 5 min<br />
|-<br />
| 1 || 8 || inf<br />
|}<br />
<br />
<br />
'''Results:'''<br />
* Amplification worked very well, we had bright bands on the right height.<br />
* Bands were cut out and DNA purified using QIAquick Gel Extration Kit.<br />
<br />
===Restriction digest of pSB4K5 (FS_01 to FS_16; 4.2 kb; [[PSB4K5#17-08-2013|17-08-2013]]) with DpnI===<br />
<br />
[[File:Heidelberg_20130818 G0 2log pSB4K5DpnI 7xOPbesch.png|150px|thumb|Restriction digest of pSB4K5 (17-08) with DpnI (17-08) and amplification of DelOP (18-08); run at 100 V, 0.8 % gel (TAE)]]<br />
[[File:Heidelberg_20130818 G0 2log pSB4K5DpnI 7xOP cutbesch.png|150px|thumb|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)]]<br />
<br />
Incubation at 37°C for about 6 hours<br />
{| class="wikitable"<br />
|-<br />
! what !! µl <br />
|-<br />
| FS_16 to FS_01 ([[PSB4K5#17-08-2013|17-08-2013]]) || 30<br />
|-<br />
| DpnI || 2<br />
|-<br />
| CutSmart Buffer || 4<br />
|-<br />
|dd H<sub>2</sub>O || 4<br />
|}<br />
<br />
'''Results:'''<br />
* Bands were cut out and DNA purified using QIAquick Gel Extration Kit.<br />
<div style="clear:both"></div><br />
<br />
==18-08-2013==<br />
<br />
<br />
===Amplification from FS_01 to FS_16; 4.2 kb===<br />
<br />
:'''Reaction''' <br />
(3x50µl)<br />
{| class="wikitable"<br />
|-<br />
! what !! µl <br />
|-<br />
| Template pSB4K5|| 1<br />
|-<br />
| FS_01: (1/10) || 2.5<br />
|-<br />
| FS_16: (1/10) || 2.5<br />
|-<br />
| Phusion Master Mix || 25<br />
|-<br />
| DMSO || 2.5<br />
|-<br />
| dd H<sub>2</sub>O || 16.5<br />
|}<br />
<br />
<br />
:'''Conditions'''<br />
{| class="wikitable"<br />
|-<br />
! colspan="3" | Biorad MyCycler*<br />
|-<br />
! Cycles-PCR !! temperature [°C] !! Time [s]<br />
|-<br />
| 1 || 98 || 5<br />
|-<br />
| rowspan="3"| 12 || 98 || 1<br />
|-<br />
| 62 || 5<br />
|-<br />
| 72 || 1:30 min<br />
|-<br />
| 1 ||72 || 5 min<br />
|-<br />
| 1 || 8 || inf<br />
|}<br />
<br />
===Restriction digest of pSB4K5 (FS_01 to FS_16; 4.2 kb; [[PSB4K5#18-08-2013|18-08-2013]]) with DpnI===<br />
<br />
Incubation at 37°C for about 6 hours<br />
{| class="wikitable"<br />
|-<br />
! what !! µl <br />
|-<br />
| FS_16 to FS_01 ([[PSB4K5#18-08-2013|18-08-2013]]) || 30<br />
|-<br />
| DpnI || 2<br />
|-<br />
| CutSmart Buffer || 4<br />
|-<br />
|dd H<sub>2</sub>O || 4<br />
|}<br />
<br />
'''Results:'''<br />
* Digested fragment was run on a gel.<br />
* Bands were cut out and DNA purified using QIAquick Gel Extration Kit.</div>
Charly