User:Karmella Haynes/Notebook/BioBrick cloning/Entry Base: Difference between revisions

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{|{{table}} width="800"
{|{{table}} width="800"
|-
|-
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Karmella's BioBrick Cloning</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}}
|-
|-
| colspan="2"|
| colspan="2"|
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
==11/18/09==
==mm/dd/yy==
* Unfinished line item
<!-- Precede finished items with a checkmark &#x2713; -->
* &#x2713; Finished line item
* Line item 1
* Line item 2
 


----
----
'''Assemblies'''
'''Line item'''
 
* Minipreps
** Sigma GenElute kit, elute w/ 75 μL elution sln.
 
* Digests
** Check with #/# digests
 
{| {{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.
|}
 
----
'''Line item'''
 
 
* 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
# 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)<br>
--> Specific notes<br>


{| class="wikitable" border="0" cellspacing="3" <!-- Digest rxn table -->
* Digests (Fermentas FD)
|-
** Specific notes
| <u>Reagent</u> || <u>Volume</u>
 
{| {{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
| DNA (plasmid) || up to 25 μL
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> Measure conc.'s
* Measure conc.'s
{| class="wikitable" border="0" cellspacing="3" <!-- [DNA] table -->
{| {{table}} cellspacing="3" <!-- [DNA] table -->
|-
|- bgcolor=#cfcfcf
| <u>Sample</u> || <u>OD260</u> || <u>260/280</u> || <u>ng/μL</u>
| Sample || OD260 || 260/280 || ng/μL
|-
| 1. KAH81 (X/P) || 0.519 || 1.67 || 25.9
|-
| 2. huPcCD (E/S) || 1.590 || 1.76 || 79.5
|-
| 3. mPcCD (E/S) || 1.604 || 1.76 || 79.5
|-
| 4. flyPcCD (E/S) || 0.903 || 1.66 || 45.2
|-
| 5. fshPcCD (E/S) || 1.482 || 1.74 || 74.1
|-
| 6. hCBX8 (E/S) || 0.691 || 1.74 || 34.5
|-
|-
| 7. flyPc (E/S) || 0.118 || 1.44 || 5.9
| 1. Digested part (a/b) || --- || --- || ---
|-
|-
| 2. KAH74 (E/X) || 0.188 || 1.37 || 9.4
| 2. Digested part (c/d) || --- || --- || ---
|}
|}




> Dephosphorylation (Roche)
* Dephosphorylation (Roche)
{| class="wikitable" border="0" cellspacing="3" <!-- Dephos table -->
{| {{table}} cellspacing="3" <!-- Dephos table -->
|-
|-
| <u>Reagent</u> || <u>Volume</u>
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
|-
|-
| DNA (clean digest) || up to 17 μL (500 ng)
| DNA (clean digest) || up to 17 μL (500 ng)
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* Ligations
# insert/(a/b)/size + vector/(c/d)/size
# vector/(c/d)/size


> Ligations (*Note: use insert + old backbone in calculations)
{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
{| class="wikitable" border="0" cellspacing="3"
|- valign="top"
| <u>Ligation</u> || <u>Plate results (lig : neg crtl)</u> 11/19/09
| bgcolor=#cfcfcf | Reagent
|-
| bgcolor=#cfcfcf | Rxn1
| 1. KAH81(X/P)/2640, 33 ng + KAH48(S/P)/~3200, 20 ng || KAH86 >10:1 (Pick 2)
| bgcolor=#cfcfcf | Rxn2
|-
|-
| 2. KAH48(S/P)/ 20 ng || &nbsp;
| Insert DNA        || ###  || ---
|-
|-
| 3. huPcCD(E/S)/3386*, > 42 ng + KAH74(E/X)/4061, 25 ng || KAH87 >10:1 (Pick 2)
| Vector DNA        || ###  || ###
|-
|-
| 4. mPcCD(E/S)/3386*, > 42 ng + " || KAH88 >10:1 (Pick 2)
| 2x lgn buf (Roche) || ###  || ###
|-
|-
| 5. flyPcCD(E/S)/3386*, > 42 ng + " || KAH89 >10:1 (Pick 2)
| T4 ligase (NEB)   || 1.0 || 1.0
|-
|-
| 6. fshPcCD(E/S)/3386*, > 42 ng + " || KAH90 >10:1 (Pick 2)
| dH<sub>2</sub>O    || ### || ###
|-
|-
| 7. hCBX8(E/S)/1170, > 42 ng + "  || KAH91 >10:1 (Pick 2)
| &nbsp;            || # μL || # μL
|-
| 8. hCBX8(E/S)/1170, > 42 ng + "  || KAH92 >10:1 (Pick 2)
|-
| 9. KAH74(S/P)/ 20 ng || &nbsp;
|}
|}
{| class="wikitable" border="0" cellspacing="3"
| &nbsp; || 1 || 2 || 3 || 4 || 5 || 6 || 7 || 8 || 9
|-
| Insert DNA        || 1.4 || --- || 1.7 || 1.7 || 1.7 || 1.7 || 0.5 || 2.5 || ---
|-
| Vector DNA        || 0.6 || 0.6 || 2.5 || 2.5 || 2.5 || 2.5 || 2.5 || 2.5 || ---
|-
| 2x lgn buf (Roche) || 5.0 || 5.0 || 5.5 || 5.5 || 5.5 || 5.5 || 5.5 || 5.5 || 5.5
|-
| T4 ligase (NEB)    || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 || 1.0
|-
| dH<sub>2</sub>O    || 2.0 || 3.4 || 0.3 || 0.3 || 0.3 || 0.3 || 1.5 || --- || 2.0
|-
| &nbsp; || 10 μL || 10 μL || 11 μL || 11 μL || 11 μL || 11 μL || 11 μL || 11.5 μL || 11 μL
|}
----
'''Oligo annealing'''
# NLS myc stop (KAH011)
# K-stop (KAH012)
# Adapter BglII/EcoRI
# Adapter PstI/BglII




{| class="wikitable" border="0" cellspacing="3"
| DNA (oligos, 100 μM) || up to 18 μL
|-
| 10x annealing buffer || 2.0
|-
| dH<sub>2</sub>O || ---
|-
| &nbsp; || 20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight
|}




Latest revision as of 17:56, 26 September 2017

Karmella's BioBrick Cloning Main project page

mm/dd/yy

  • Line item 1
  • Line item 2


Line item

  • Minipreps
    • Sigma GenElute kit, elute w/ 75 μL elution sln.
  • Digests
    • Check with #/# digests
Reagent Volume Expected:
1. BB 1 = size
2. BB2 = size
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 9.5
  15 μL --> 37°C/ ~15 min.

Line item


  • Assemblies
  1. BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
  2. BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size


  • Digests (Fermentas FD)
    • Specific notes
Reagent Volume
DNA (plasmid) up to 25 μL
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O ---
  30 μL --> 37°C/ ~30 min.


  • Measure conc.'s
Sample OD260 260/280 ng/μL
1. Digested part (a/b) --- --- ---
2. Digested part (c/d) --- --- ---


  • Dephosphorylation (Roche)
Reagent Volume
DNA (clean digest) up to 17 μL (500 ng)
10x buffer d.p. 2.0
phosphatase 1.0
dH2O ---
  20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL


  • Ligations
  1. insert/(a/b)/size + vector/(c/d)/size
  2. vector/(c/d)/size
Reagent Rxn1 Rxn2
Insert DNA ### ---
Vector DNA ### ###
2x lgn buf (Roche) ### ###
T4 ligase (NEB) 1.0 1.0
dH2O ### ###
  # μL # μL




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