User:Karmella Haynes/Notebook/BioBrick cloning/2013/03/06: Difference between revisions

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==mm/dd/yy==
==03/06/13==
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* Line item 1
* Colony PCR: for Rene's project
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----
----
'''Minipreps'''<br>
'''Colony PCR'''<br>
* Check with E/P digests
* Streaked single colonies on gridded plates yesterday
* Used bit of streak for PCR today
* All parts are on pSB1A3-based vectors (http://partsregistry.org/Part:pSB1A3)
** Use primers VF2 and VR to amplify inserts


{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
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| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume  
| bgcolor=#cfcfcf | Volume  
| rowspan="7" | <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br>
| rowspan="7" | <u>Expected:</u><br>Primers add ~250bp<br>1-3. K084007 (835 bp) = 1085<br>4-6. R0071 (53 bp) = 303<br>7. K081016 (735 bp) = 985
| 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] -->
| rowspan="7" | [[Image:KAH030713_gel1.jpg|300px|PCR gel]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
|-
| DNA(plasmid) || 2.0 μL
| Bacteria from tip || ---
|-
|-
| 10X buffer || 1.5
| 10 μM VF2 primer || 1.0
|-
|-
| EcoRI || 1.0
| 10 μM VR primer  || 1.0
|-
|-
| PstI || 1.0
| 2x GoTaq Green || 12.5
|-
|-
| dH<sub>2</sub>O || 9.5
| dH<sub>2</sub>O || 10.5
|-
|-
| &nbsp; || 15 μL --> 37°C/ ~15 min.
| &nbsp; || 25.0
|}
 
----
'''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
|}
|}


PCR
* 95°C, 3 min
* [95°C, 15 sec; 57°C 15 sec; 72 °C 60 sec] x35
* 72°C, 3 min
* 4°C, ∞


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Revision as of 18:18, 7 March 2013

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03/06/13

  • Colony PCR: for Rene's project


Colony PCR

  • Streaked single colonies on gridded plates yesterday
  • Used bit of streak for PCR today
  • All parts are on pSB1A3-based vectors (http://partsregistry.org/Part:pSB1A3)
    • Use primers VF2 and VR to amplify inserts
Reagent Volume Expected:
Primers add ~250bp
1-3. K084007 (835 bp) = 1085
4-6. R0071 (53 bp) = 303
7. K081016 (735 bp) = 985 		PCR gel
10 μL/lane; 1% agarose; Ladder
Bacteria from tip ---
10 μM VF2 primer 1.0
10 μM VR primer 1.0
2x GoTaq Green 12.5
dH2O 10.5
  25.0

PCR

  • 95°C, 3 min
  • [95°C, 15 sec; 57°C 15 sec; 72 °C 60 sec] x35
  • 72°C, 3 min
  • 4°C, ∞


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