User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/05: Difference between revisions

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(Autocreate 2013/01/05 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning)
 
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==mm/dd/yy==
==01/05/13==
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* Line item 1
* Golden gate: PCR parts, complete protocol for Brady's construct
* Line item 2
* Gibson: PCR parts, complete protocol for Brady's construct




----
----
'''Minipreps'''<br>
'''Golden Gate Assembly'''<br>
* Check with E/P digests
* Try new protocol from Dave Savage
# hPCD + BL01 + V0120
# hPCD + BL01 + pSB1A3
 
* Check the sequences for BsmBI sites
 
'''PCR'''
# V0120, gg_? / gg_?
# pSB1A3, gg_? / gg_?
# hPCD, gg_BBP-hPCD F  / gg_hPCD R
# BL01, gg_BL01 F / gg_BL01-BBS R


{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
Line 20: Line 30:
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume  
| bgcolor=#cfcfcf | Volume  
| rowspan="7" | <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br>
| rowspan="7" | <u>Expected:</u><br>1. V0120 = size<br>2. pSB1A3 = size<br>3. hPCD = size<br>4. BL01 = size
| 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] -->
| 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
| DNA(plasmid) || 0.5 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"
| 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
| 10 μM primer 1 || 1.0
|-
|-
| dH<sub>2</sub>O || ---
| 10 μM primer 2 || 1.0
|-
|-
| &nbsp; || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
| 2x GoTaq mix || 25.0
|}
 
 
* 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>
| dH<sub>2</sub>O || ##
|-
|-
| 2. vector(c/d)/ ## ng || &nbsp;
| &nbsp; || 50 μL
|}
|}


{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
* 95°C, 3 min.
| &nbsp;             || 1    || 2   ||
* [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 2 min.] x 30
|-
* 72°C, 3 min.
| Insert DNA        || ###  || ---  ||
* 4°C, ∞
|-
| 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 11:30, 5 January 2013

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01/05/13

  • Golden gate: PCR parts, complete protocol for Brady's construct
  • Gibson: PCR parts, complete protocol for Brady's construct


Golden Gate Assembly

  • Try new protocol from Dave Savage
  1. hPCD + BL01 + V0120
  2. hPCD + BL01 + pSB1A3
  • Check the sequences for BsmBI sites

PCR

  1. V0120, gg_? / gg_?
  2. pSB1A3, gg_? / gg_?
  3. hPCD, gg_BBP-hPCD F / gg_hPCD R
  4. BL01, gg_BL01 F / gg_BL01-BBS R
Reagent Volume Expected:
1. V0120 = size
2. pSB1A3 = size
3. hPCD = size
4. BL01 = size
DNA(plasmid) 0.5 μL
10 μM primer 1 1.0
10 μM primer 2 1.0
2x GoTaq mix 25.0
dH2O ##
  50 μL
  • 95°C, 3 min.
  • [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 2 min.] x 30
  • 72°C, 3 min.
  • 4°C, ∞




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