User:Karmella Haynes/Notebook/BioBrick cloning/2015/01/27: Difference between revisions

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==mm/dd/yy==
==01/27/15==
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* Line item 1
* Gel purification - KAH60 vector PCR, ATF2 PCR
* Line item 2
* PCR - re-amplify KAH 60 vector
* Review LCR procedure from Cameron: http://openwetware.org/wiki/Haynes_Lab:Notebook/Engineering_PC-TFs/2014/12/22




----
----
'''Minipreps'''<br>
'''Gel purification'''<br>
* Check with E/P digests
* Gel purify fragments from PCR done on [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2015/01/22 01/22/15]
 
* KAH60/pcVN - 6637 bp
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
* ATF2 - 960 bp
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
| 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


'''PCR'''
* Use purified KAH60 fragments as template for PCR
# KAH60 "backbone" (without VP64, includes scars) = 6637: KAH60 F1/ KAH60 R1


* 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 | Rxn1
| 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] -->
| rowspan="7" | Expected:<br>1. KAH60 = 6637
| rowspan="7" | [[Image:KAH012715_gel.jpg|270px|Hover name]]<br>10 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
|-
| DNA (plasmid) || up to 25 μL
| Template || 1.0
|-
|-
| 10x buffer || 3.0
| 10 uM fwd primer || 1.0
|-
|-
| enzyme 1 || 1.0
| 10 uM rev primer || 1.0
|-
|-
| enzyme 2 || 1.0
| 2x GoTaq green || 12.5
|-
|-
| dH<sub>2</sub>O || ---
| dH<sub>2</sub>O || 9.5
|-
| &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) || --- || --- || ---
| &nbsp; || 25.0
|}
|}


Program: GOTAQ35cyc
* 95°C, 3 min
* 30x[95°C, 1 min; 57°C, 1 min; 72°C, 3 min]
* 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 16:07, 27 January 2015

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01/27/15


Gel purification

  • Gel purify fragments from PCR done on 01/22/15
  • KAH60/pcVN - 6637 bp
  • ATF2 - 960 bp

PCR

  • Use purified KAH60 fragments as template for PCR
  1. KAH60 "backbone" (without VP64, includes scars) = 6637: KAH60 F1/ KAH60 R1


Reagent Rxn1 Expected:
1. KAH60 = 6637 		Hover name
10 μL/lane, 1% agarose; Ladder
Template 1.0
10 uM fwd primer 1.0
10 uM rev primer 1.0
2x GoTaq green 12.5
dH2O 9.5
  25.0

Program: GOTAQ35cyc

  • 95°C, 3 min
  • 30x[95°C, 1 min; 57°C, 1 min; 72°C, 3 min]
  • 72°C, 3 min
  • 4°C ∞



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