Difference between revisions of "User:Karmella Haynes/Notebook/BioBrick cloning/2013/03/05"

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(Autocreate 2013/03/05 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning)
 
(03/05/13)
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| colspan="2"|
 
| colspan="2"|
 
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
 
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
==mm/dd/yy==
+
==03/05/13==
 
<!-- Precede finished items with a checkmark &#x2713; -->
 
<!-- Precede finished items with a checkmark &#x2713; -->
* Line item 1
+
* Colony PCR: streak libraries from Rene's transformation plates
* Line item 2
 
  
  
 
----
 
----
'''Minipreps'''<br>
+
'''Colony PCR: streak library'''<br>
* Check with E/P digests
+
* Pick and streak three colonies from each plate
 +
* LB agar amp 100 μg/mL
  
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
+
Plate 1
|- valign="top"
+
* 1-1..3: BBa_K092900, http://partsregistry.org/Part:BBa_K092900
| bgcolor=#cfcfcf | Reagent
+
* 2-1..3: BBa_K092100, http://partsregistry.org/Part:BBa_K092100
| bgcolor=#cfcfcf | Volume
+
* 3-1..3: BBa_K091134, http://partsregistry.org/Part:BBa_K091134
| rowspan="7" | <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br>
+
* 4-1..3: BBa_K145254, http://partsregistry.org/Part:BBa_K145254
| rowspan="7" | <!-- [[Image:GelImage.jpg|400px|Hover name]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] -->
+
* 5-1..3: BBa_K081023, http://partsregistry.org/Part:BBa_K081023
|-
+
* 6-1..3: BBa_F2620, http://partsregistry.org/Part:BBa_F2620
| DNA(plasmid) || 2.0 μL
+
* 7-1..3: BBa_K084007, http://partsregistry.org/Part:BBa_K084007
|-
+
* 8-1..3: BBa_R0071, http://partsregistry.org/Part:BBa_R0071
| 10X buffer || 1.5
+
* 9-1..3: BBa_K082035, http://partsregistry.org/Part:BBa_K082035
|-
+
* 10-1..3: BBa_K081016, http://partsregistry.org/Part:BBa_K081016
| EcoRI || 1.0
 
|-
 
| PstI || 1.0
 
|-
 
| dH<sub>2</sub>O || 9.5
 
|-
 
| &nbsp; || 15 μL --> 37°C/ ~15 min.
 
|}
 
  
----
 
'''Assemblies'''
 
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
 
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
 
 
 
* Digests (Fermentas FD)
 
** Specific notes
 
 
{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
 
|- valign="top"
 
| bgcolor=#cfcfcf | Reagent
 
| bgcolor=#cfcfcf | Volume
 
| 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] -->
 
|-
 
| DNA (plasmid) || up to 25 μL
 
|-
 
| 10x buffer || 3.0
 
|-
 
| enzyme 1 || 1.0
 
|-
 
| enzyme 2 || 1.0
 
|-
 
| dH<sub>2</sub>O || ---
 
|-
 
| &nbsp; || 30 μL --> 37°C/ ~30 min.
 
|}
 
 
 
* Measure conc.'s
 
{| {{table}} cellspacing="3" <!-- [DNA] table -->
 
|- bgcolor=#cfcfcf
 
| Sample || OD260 || 260/280 || ng/μL
 
|-
 
| 1. Digested part (a/b) || --- || --- || ---
 
|-
 
| 2. Digested part (c/d) || --- || --- || ---
 
|}
 
 
 
* Dephosphorylation (Roche)
 
{| {{table}} cellspacing="3" <!-- Dephos table -->
 
|-
 
| bgcolor=#cfcfcf | Reagent
 
| bgcolor=#cfcfcf | Volume
 
|-
 
| DNA (clean digest) || up to 17 μL (500 ng)
 
|-
 
| 10x buffer d.p. || 2.0
 
|-
 
| phosphatase || 1.0
 
|-
 
| dH<sub>2</sub>O || ---
 
|-
 
| &nbsp; || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
 
|}
 
 
 
* Ligations
 
{| {{table}} cellspacing="3" <!-- Ligations table -->
 
|- bgcolor=#cfcfcf
 
| Ligation || <font color="blue"><u>Plate results (lig : neg crtl)</u> mm/dd/yy</font>
 
|-
 
| 1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng || <font color="blue">new BioBrick #:1 (Pick #)</font>
 
|-
 
| 2. vector(c/d)/ ## ng || &nbsp;
 
|}
 
 
{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
 
| &nbsp;            || 1    || 2    ||
 
|-
 
| Insert DNA        || ###  || ---  ||
 
|-
 
| Vector DNA        || ###  || ###  ||
 
|-
 
| 2x lgn buf (Roche) || ###  || ###  ||
 
|-
 
| T4 ligase (NEB)    || 1.0  || 1.0  ||
 
|-
 
| dH<sub>2</sub>O    || ###  || ###  ||
 
|-
 
| &nbsp;            || # μL || # μL ||
 
|}
 
 
----
 
'''Oligo annealing'''
 
# New BB 1
 
# New BB 2
 
 
{| class="wikitable" border="0" cellspacing="3" <!-- Oligo annealing rxn table -->
 
| DNA (oligos, 100 μM) || up to 18 μL (3 μL ea.)
 
|-
 
| 10x annealing buffer || 2.0
 
|-
 
| dH<sub>2</sub>O || ---
 
|-
 
| &nbsp; || 20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight
 
|}
 
  
  

Revision as of 17:15, 7 March 2013

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03/05/13

  • Colony PCR: streak libraries from Rene's transformation plates



Colony PCR: streak library

  • Pick and streak three colonies from each plate
  • LB agar amp 100 μg/mL

Plate 1