Difference between revisions of "User:Karmella Haynes/Notebook/BioBrick cloning/2013/04/12"

From OpenWetWare
Jump to: navigation, search
(04/12/13)
(fix raw html notebook nav)
 
(5 intermediate revisions by one other user not shown)
Line 2: Line 2:
 
|-
 
|-
 
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Karmella's BioBrick Cloning</span>
 
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Karmella's BioBrick Cloning</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
+
|style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}}
 
|-
 
|-
 
| colspan="2"|
 
| colspan="2"|
Line 15: Line 15:
  
 
'''MV9 building: colony PCR'''
 
'''MV9 building: colony PCR'''
* Pick 8 colonies from plates 1, 2, 3, and 4; make a streak plate
+
* Pick 8 colonies from plates 1, 2, 3, and 4; make a mini-streak library, then wash pipette tip in PCR master mix
* Pick 4 colonies from plate 5 (neg. ctrl)
+
* Pick 4 colonies from plate 5 (neg. ctrl); do not add to streak library; include in PCR as neg ctrl.s
 
* Primers for MV2 vector: DD122B (fwd), DD123B (rev); see [http://partsregistry.org/Part:BBa_J176122 BBa_J176122]
 
* Primers for MV2 vector: DD122B (fwd), DD123B (rev); see [http://partsregistry.org/Part:BBa_J176122 BBa_J176122]
  
Line 23: Line 23:
 
| bgcolor=#cfcfcf | Reagent
 
| bgcolor=#cfcfcf | Reagent
 
| bgcolor=#cfcfcf | Volume
 
| bgcolor=#cfcfcf | Volume
 +
| bgcolor=#cfcfcf | x38
 
| rowspan="7" | Expected:<br>MV2 (empty) = 177<br>MV9 (single insert) = 253<br>extra inserts = +76
 
| rowspan="7" | Expected:<br>MV2 (empty) = 177<br>MV9 (single insert) = 253<br>extra inserts = +76
| rowspan="7" | <!-- [[Image:GelImage.jpg|270px|Hover name]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] -->
+
| rowspan="7" | [[Image:KAH041513_gel1.jpg|270px|Hover name]]<br>10 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
 
|-
 
|-
| DNA (colony) || ---
+
| DNA (colony) || --- || ---
 
|-
 
|-
| 10 μM primer 1 || 1.0
+
| 10 μM primer 1 || 1.0 || 38.0
 
|-
 
|-
| 10 μM primer 2 || 1.0
+
| 10 μM primer 2 || 1.0 || 38.0
 
|-
 
|-
| 2x GoTaq green || 10.0
+
| 2x GoTaq green || 10.0 || 380.0
 
|-
 
|-
| dH<sub>2</sub>O || 8.0
+
| dH<sub>2</sub>O || 8.0 || 304.0
 
|-
 
|-
| &nbsp; || 20 μL
+
| &nbsp; || 20 μL || &nbsp;
 
|}
 
|}
  

Latest revision as of 21:37, 26 September 2017

Owwnotebook icon.pngKarmella's BioBrick Cloning Report.pngMain project page
Resultset previous.pngPrevious entry      Next entryResultset next.png

04/12/13

  • Chem. Competent cell prep: CaCl wash; 15% glycerol/CaCl resuspension (used 30 mL instead of 20 mL); incubate in cold room on ice o/n
  • MV9 building: colony PCR



MV9 building: colony PCR

  • Pick 8 colonies from plates 1, 2, 3, and 4; make a mini-streak library, then wash pipette tip in PCR master mix
  • Pick 4 colonies from plate 5 (neg. ctrl); do not add to streak library; include in PCR as neg ctrl.s
  • Primers for MV2 vector: DD122B (fwd), DD123B (rev); see BBa_J176122
Reagent Volume x38 Expected:
MV2 (empty) = 177
MV9 (single insert) = 253
extra inserts = +76
Hover name
10 μL/lane, 1% agarose; Ladder
DNA (colony) --- ---
10 μM primer 1 1.0 38.0
10 μM primer 2 1.0 38.0
2x GoTaq green 10.0 380.0
dH2O 8.0 304.0
  20 μL  

Thermal cycling

  • 95°C, 3 min.
  • [95°C, 15 sec; 57°C, 15 sec; 72°C, 15 sec] x30
  • 72°C, 3 min.
  • 4°C, ∞