User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/19: Difference between revisions

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<!-- Precede finished items with a checkmark &#x2713; -->
<!-- Precede finished items with a checkmark &#x2713; -->
* Cas-tone - cultures, minipreps, digests
* Cas-tone - cultures, minipreps, digests
* Rene - PCR library 96-well plates
* Rene - PCR library 96-well plate




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--> 37°C/ ~10 min.
--> 37°C/ ~10 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'''
'''Rene - PCR libraries'''
# New BB 1
* Inoculate 200 μL mini-cultures (37°C) with individual colonies picked from PCR cloning plates with sterile micropipette tips
# New BB 2
# Luc14g034: 192 colonies, 2 96-well plates (A and B)
# Gal4EEDg034: 192 colonies, 2 96-well plates (A and B)
* Spot cultures from 96-well plates onto LB agar amp (100μg/mL) using multichannel pipettor
** Transferred 2 μL from each well to a 6x8 "array"
** Each 96-well set was spotted onto two plates (A1-G1, A7-G7)
** 8 plates total


{| 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 14:56, 21 May 2015

Karmella's BioBrick Cloning <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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05/19/15

  • Cas-tone - cultures, minipreps, digests
  • Rene - PCR library 96-well plate


Cas-tone - cultures, minipreps, digests

  • Minipreps
    • Check with FD E/P digests
    • Modification (dsOligo insert replacing VP64) removes an EcoRI site
Reagent Volume Expected:
1,2. CMV-dCas9-HA = 5317, 4189
CMV-dCas9-VP64 =5459, 3016, 1337
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 9.5
  15 μL

--> 37°C/ ~10 min.


Rene - PCR libraries

  • Inoculate 200 μL mini-cultures (37°C) with individual colonies picked from PCR cloning plates with sterile micropipette tips
  1. Luc14g034: 192 colonies, 2 96-well plates (A and B)
  2. Gal4EEDg034: 192 colonies, 2 96-well plates (A and B)
  • Spot cultures from 96-well plates onto LB agar amp (100μg/mL) using multichannel pipettor
    • Transferred 2 μL from each well to a 6x8 "array"
    • Each 96-well set was spotted onto two plates (A1-G1, A7-G7)
    • 8 plates total



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