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

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==mm/dd/yy==
==04/12/13==
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* Line item 1
* 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
* Line item 2
* MV9 building: colony PCR




----
----
'''Minipreps'''<br>
* Check with E/P digests


{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
'''MV9 building: colony PCR'''
|- valign="top"
* Pick 8 colonies from plates 1, 2, 3, and 4; make a mini-streak library, then wash pipette tip in PCR master mix
| bgcolor=#cfcfcf | Reagent
* Pick 4 colonies from plate 5 (neg. ctrl); do not add to streak library; include in PCR as neg ctrl.s
| bgcolor=#cfcfcf | Volume
* Primers for MV2 vector: DD122B (fwd), DD123B (rev); see [http://partsregistry.org/Part:BBa_J176122 BBa_J176122]
| rowspan="7" | <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br>
| 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] -->
|-
| DNA(plasmid) || 2.0 μL
|-
| 10X buffer || 1.5
|-
| 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 -->
{| {{table}} cellspacing="3" <!-- PCR rxn. table -->
|- valign="top"
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
| 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] -->
| bgcolor=#cfcfcf | x38
|-
| rowspan="7" | Expected:<br>MV2 (empty) = 177<br>MV9 (single insert) = 253<br>extra inserts = +76
| DNA (plasmid) || up to 25 μL
| 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]
|-
| 10x buffer || 3.0
|-
|-
| enzyme 1 || 1.0
| DNA (colony) || --- || ---
|-
|-
| enzyme 2 || 1.0
| 10 μM primer 1 || 1.0 || 38.0
|-
|-
| dH<sub>2</sub>O || ---
| 10 μM primer 2 || 1.0 || 38.0
|-
|-
| &nbsp; || 30 μL --> 37°C/ ~30 min.
| 2x GoTaq green || 10.0 || 380.0
|}
 
 
* Measure conc.'s
{| {{table}} cellspacing="3" <!-- [DNA] table -->
|- bgcolor=#cfcfcf
| Sample || OD260 || 260/280 || ng/μL
|-
|-
| 1. Digested part (a/b) || --- || --- || ---
| dH<sub>2</sub>O || 8.0 || 304.0
|-
|-
| 2. Digested part (c/d) || --- || --- || ---
| &nbsp; || 20 μL || &nbsp;
|}
|}


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, ∞


* 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 16:11, 19 April 2013

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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, ∞



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