User:Karmella Haynes/Notebook/BioBrick cloning/2013/03/06

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(Autocreate 2013/03/06 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning)
Current revision (21:18, 7 March 2013) (view source)
(→03/06/13)
 
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==mm/dd/yy==
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==03/06/13==
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* Line item 1
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* Colony PCR: for Rene's project
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* Line item 2
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----
----
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'''Minipreps'''<br>
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'''Colony PCR'''<br>
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* Check with E/P digests
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* 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  
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| rowspan="7" | <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br>
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| 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
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| 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] -->
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| 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]
|-
|-
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| DNA(plasmid) || 2.0 μL
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| Bacteria from tip || ---
|-
|-
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| 10X buffer || 1.5
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| 10 μM VF2 primer || 1.0
|-
|-
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| EcoRI || 1.0
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| 10 μM VR primer  || 1.0
|-
|-
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| PstI || 1.0
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| 2x GoTaq Green || 12.5
|-
|-
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| dH<sub>2</sub>O || 9.5
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| dH<sub>2</sub>O || 10.5
|-
|-
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| &nbsp; || 15 μL --> 37°C/ ~15 min.
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| &nbsp; || 25.0
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|}
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----
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'''Assemblies'''
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# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
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# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
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* Digests (Fermentas FD)
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** Specific notes
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{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
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|- valign="top"
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| bgcolor=#cfcfcf | Reagent
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| bgcolor=#cfcfcf | Volume
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| 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] -->
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|-
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| DNA (plasmid) || up to 25 μL
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|-
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| 10x buffer || 3.0
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|-
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| enzyme 1 || 1.0
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|-
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| enzyme 2 || 1.0
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|-
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| dH<sub>2</sub>O || ---
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|-
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| &nbsp; || 30 μL --> 37°C/ ~30 min.
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|}
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* Measure conc.'s
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{| {{table}} cellspacing="3" <!-- [DNA] table -->
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|- bgcolor=#cfcfcf
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| Sample || OD260 || 260/280 || ng/μL
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|-
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| 1. Digested part (a/b) || --- || --- || ---
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|-
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| 2. Digested part (c/d) || --- || --- || ---
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|}
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* Dephosphorylation (Roche)
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{| {{table}} cellspacing="3" <!-- Dephos table -->
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|-
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| bgcolor=#cfcfcf | Reagent
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| bgcolor=#cfcfcf | Volume
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|-
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| DNA (clean digest) || up to 17 μL (500 ng)
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|-
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| 10x buffer d.p. || 2.0
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|-
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| phosphatase || 1.0
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|-
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| dH<sub>2</sub>O || ---
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|-
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| &nbsp; || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
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|}
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* Ligations
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{| {{table}} cellspacing="3" <!-- Ligations table -->
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|- bgcolor=#cfcfcf
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| Ligation || <font color="blue"><u>Plate results (lig : neg crtl)</u> mm/dd/yy</font>
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|-
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| 1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng || <font color="blue">new BioBrick #:1 (Pick #)</font>
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|-
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| 2. vector(c/d)/ ## ng || &nbsp;
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|}
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{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
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| &nbsp;            || 1    || 2    ||
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|-
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| Insert DNA        || ###  || ---  ||
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|-
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| Vector DNA        || ###  || ###  ||
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|-
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| 2x lgn buf (Roche) || ###  || ###  ||
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|-
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| T4 ligase (NEB)    || 1.0  || 1.0  ||
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|-
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| dH<sub>2</sub>O    || ###  || ###  ||
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|-
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| &nbsp;            || # μL || # μL ||
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|}
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----
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'''Oligo annealing'''
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# New BB 1
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# New BB 2
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{| class="wikitable" border="0" cellspacing="3" <!-- Oligo annealing rxn table -->
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| DNA (oligos, 100 μM) || up to 18 μL (3 μL ea.)
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|-
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| 10x annealing buffer || 2.0
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|-
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| dH<sub>2</sub>O || ---
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|-
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| &nbsp; || 20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight
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|}
|}
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PCR
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* 95°C, 3 min
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* [95°C, 15 sec; 57°C 15 sec; 72 °C 60 sec] x35
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* 72°C, 3 min
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* 4°C, ∞
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->

Current revision

Karmella's BioBrick Cloning Main project page
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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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