User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/07: Difference between revisions

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----
'''LCR - test 5'-phos primers'''<br>
'''LCR - test 5'-phos primers'''<br>
# Gal4DB-mCh (459 bp): Template - KAH228 plasmid, F - Gal4DB f1, R - mCh r1
# '''Gal4DB-mCh''' (459 bp): Template - KAH228 plasmid, F - Gal4DB f1, R - mCh r1
# ATF2: Template - iPSC cDNA (Brafman), F - ATF2_f1, R - ATF2_r1
# '''ATF2''' (906 bp): Template - iPSC cDNA (Brafman), F - ATF2_f1, R - ATF2_r1
# MV10 (backbone): Template - MV10 plasmid, F - MV10 f1, R - MV10 r1
# (wait for primers to arrive) '''MV10''' (backbone): Template - MV10 plasmid, F - MV10 f1, R - MV10 r1


 
{| {{table}} cellspacing="3" <!-- PCR rxn. table -->
 
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
|- 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
 
 
* Digests (Fermentas FD)
** Specific notes
 
{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
|- valign="top"
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
| bgcolor=#cfcfcf | Rxn1,2
| 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 | Rxn3,4
|-
| rowspan="7" | Expected:<br>1,2. Gal4DB-mCh = 459<br>3,4. ATF2 = 906
| DNA (plasmid) || up to 25 μL
| rowspan="7" | [[Image:somegel.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
|-
| 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        || ###  || ---  ||
| Template || 0.2 || 1.0 || 1.0 || 1.0
|-
|-
| Vector DNA        || ###  || ###  ||
| 10 uM fwd primer || 1.0 || 1.0 || 1.0 || 1.0
|-
|-
| 2x lgn buf (Roche) || ###  || ###  ||
| 10 uM rev primer || 1.0 || 1.0 || 1.0 || 1.0
|-
|-
| T4 ligase (NEB)    || 1.0  || 1.0  ||
| 2x GoTaq green || 12.5 || 12.5 || 12.5 || 12.5
|-
|-
| dH<sub>2</sub>O   || ###  || ###  ||
| dH<sub>2</sub>O || 10.3 || 9.5 || 9.5 || 9.5
|-
|-
| &nbsp;             || # μL || # μL ||
| &nbsp; || 25.0 || 25.0 || 25.0 || 25.0
|}
|}


----
Program: GOTAQ35cyc
'''Oligo annealing'''
* 95°C, 3 min
# New BB 1
* 30x[95°C, 1 min; 57°C, 1 min; 72°C, 3 min]
# New BB 2
* 72°C, 3 min
 
* 4°C ∞
{| 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 17:57, 7 April 2015

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04/07/15

  • LCR development - test 5'-phos primers for Gal4DB-mCh-ATF2_MV10 assembly


LCR - test 5'-phos primers

  1. Gal4DB-mCh (459 bp): Template - KAH228 plasmid, F - Gal4DB f1, R - mCh r1
  2. ATF2 (906 bp): Template - iPSC cDNA (Brafman), F - ATF2_f1, R - ATF2_r1
  3. (wait for primers to arrive) MV10 (backbone): Template - MV10 plasmid, F - MV10 f1, R - MV10 r1
Reagent Rxn1,2 Rxn3,4 Expected:
1,2. Gal4DB-mCh = 459
3,4. ATF2 = 906 		Hover name
10 μL/lane, 1% agarose; Ladder
Template 0.2 1.0 1.0 1.0
10 uM fwd primer 1.0 1.0 1.0 1.0
10 uM rev primer 1.0 1.0 1.0 1.0
2x GoTaq green 12.5 12.5 12.5 12.5
dH2O 10.3 9.5 9.5 9.5
  25.0 25.0 25.0 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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