User:Karmella Haynes/Notebook/BioBrick cloning/Entry Base

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{|{{table}} width="800"
{|{{table}} width="800"
|-
|-
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
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|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"|<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>}}
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| 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. ##### -->
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==11/18/09==
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==mm/dd/yy==
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* Unfinished line item
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<!-- Precede finished items with a checkmark &#x2713; -->
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* &#x2713; Finished line item
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* Line item 1
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* Line item 2
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 +
----
 +
'''Minipreps'''<br>
 +
* Check with E/P digests
 +
 
 +
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
 +
|- valign="top"
 +
| bgcolor=#cfcfcf | Reagent
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| bgcolor=#cfcfcf | Volume
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| 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] -->
 +
|-
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| DNA(plasmid) || 2.0 μL
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|-
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| 10X buffer || 1.5
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|-
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| EcoRI || 1.0
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|-
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| PstI || 1.0
 +
|-
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| dH<sub>2</sub>O || 9.5
 +
|-
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| &nbsp; || 15 μL --> 37°C/ ~15 min.
 +
|}
----
----
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# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
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> Digests (Fermentas FD)<br>
 
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--> Specific notes<br>
 
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{| class="wikitable" border="0" cellspacing="3" <!-- Digest rxn table -->
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* Digests (Fermentas FD)
-
|-
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** Specific notes
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| <u>Reagent</u> || <u>Volume</u>
+
 
 +
{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
 +
|- valign="top"
 +
| bgcolor=#cfcfcf | Reagent
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| bgcolor=#cfcfcf | Volume
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| 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
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* Measure conc.'s
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{| class="wikitable" border="0" cellspacing="3" <!-- [DNA] table -->
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{| {{table}} cellspacing="3" <!-- [DNA] table -->
 +
|- bgcolor=#cfcfcf
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| Sample || OD260 || 260/280 || ng/μL
|-
|-
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| <u>Sample</u> || <u>OD260</u> || <u>260/280</u> || <u>ng/μL</u>
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| 1. Digested part (a/b) || --- || --- || ---
|-
|-
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| 1. KAH81 (X/P) || 0.519 || 1.67 || 25.9
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| 2. Digested part (c/d) || --- || --- || ---
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|-
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| 2. huPcCD (E/S) || 1.590 || 1.76 || 79.5
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-
|-
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| 3. mPcCD (E/S) || 1.604 || 1.76 || 79.5
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-
|-
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| 4. flyPcCD (E/S) || 0.903 || 1.66 || 45.2
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-
|-
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| 5. fshPcCD (E/S) || 1.482 || 1.74 || 74.1
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-
|-
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| 6. hCBX8 (E/S) || 0.691 || 1.74 || 34.5
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-
|-
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| 7. flyPc (E/S) || 0.118 || 1.44 || 5.9
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-
|-
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| 2. KAH74 (E/X) || 0.188 || 1.37 || 9.4
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|}
|}
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> Dephosphorylation (Roche)
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* Dephosphorylation (Roche)
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{| class="wikitable" border="0" cellspacing="3" <!-- Dephos table -->
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{| {{table}} cellspacing="3" <!-- Dephos table -->
|-
|-
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| <u>Reagent</u> || <u>Volume</u>
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| 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
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> Ligations (*Note: use insert + old backbone in calculations)
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{| {{table}} cellspacing="3" <!-- Ligations table -->
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{| class="wikitable" border="0" cellspacing="3"
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|- bgcolor=#cfcfcf
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| <u>Ligation</u> || <u>Plate results (lig : neg crtl)</u> 11/19/09
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| Ligation || <font color="blue"><u>Plate results (lig : neg crtl)</u> mm/dd/yy</font>
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|-
+
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| 1. KAH81(X/P)/2640, 33 ng + KAH48(S/P)/~3200, 20 ng || KAH86 >10:1 (Pick 2)
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-
|-
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| 2. KAH48(S/P)/ 20 ng || &nbsp;
+
-
|-
+
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| 3. huPcCD(E/S)/3386*, > 42 ng + KAH74(E/X)/4061, 25 ng || KAH87 >10:1 (Pick 2)
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|-
+
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| 4. mPcCD(E/S)/3386*, > 42 ng + " || KAH88 >10:1 (Pick 2)
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|-
+
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| 5. flyPcCD(E/S)/3386*, > 42 ng + "  || KAH89 >10:1 (Pick 2)
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|-
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| 6. fshPcCD(E/S)/3386*, > 42 ng + "  || KAH90 >10:1 (Pick 2)
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|-
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| 7. hCBX8(E/S)/1170, > 42 ng + "  || KAH91 >10:1 (Pick 2)
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|-
|-
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| 8. hCBX8(E/S)/1170, > 42 ng + || KAH92 >10:1 (Pick 2)
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| 1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng || <font color="blue">new BioBrick #:1 (Pick #)</font>
|-
|-
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| 9. KAH74(S/P)/ 20 ng || &nbsp;
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| 2. vector(c/d)/ ## ng || &nbsp;
|}
|}
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{| class="wikitable" border="0" cellspacing="3"
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{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
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| &nbsp; || 1 || 2 || 3 || 4 || 5 || 6 || 7 || 8 || 9
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| &nbsp;             || 1   || 2   ||
|-
|-
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| Insert DNA        || 1.4 || --- || 1.7 || 1.7 || 1.7 || 1.7 || 0.5 || 2.5 || ---
+
| Insert DNA        || ###  || --- ||
|-
|-
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| Vector DNA        || 0.6 || 0.6 || 2.5 || 2.5 || 2.5 || 2.5 || 2.5 || 2.5 || ---
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| Vector DNA        || ###  || ###  ||
|-
|-
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| 2x lgn buf (Roche) || 5.0 || 5.0 || 5.5 || 5.5 || 5.5 || 5.5 || 5.5 || 5.5 || 5.5
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| 2x lgn buf (Roche) || ###  || ###  ||
|-
|-
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| T4 ligase (NEB)    || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 || 1.0
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| T4 ligase (NEB)    || 1.0 || 1.0 ||
|-
|-
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| dH<sub>2</sub>O    || 2.0 || 3.4 || 0.3 || 0.3 || 0.3 || 0.3 || 1.5 || --- || 2.0
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| dH<sub>2</sub>O    || ###  || ###  ||
|-
|-
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| &nbsp; || 10 μL || 10 μL || 11 μL || 11 μL || 11 μL || 11 μL || 11 μL || 11.5 μL || 11 μL
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| &nbsp;             || # μL || # μL ||
|}
|}
----
----
'''Oligo annealing'''
'''Oligo annealing'''
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# NLS myc stop (KAH011)
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# New BB 1
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# K-stop (KAH012)
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# New BB 2
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# Adapter BglII/EcoRI
+
-
# Adapter PstI/BglII
+
-
 
+
-
{| class="wikitable" border="0" cellspacing="3"
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{| class="wikitable" border="0" cellspacing="3" <!-- Oligo annealing rxn table -->
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| DNA (oligos, 100 μM) || up to 18 μL
+
| DNA (oligos, 100 μM) || up to 18 μL (3 μL ea.)
|-
|-
| 10x annealing buffer || 2.0
| 10x annealing buffer || 2.0

Current revision

Karmella's BioBrick Cloning Main project page

mm/dd/yy

  • Line item 1
  • Line item 2



Minipreps

  • Check with E/P 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.

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
Ligation Plate results (lig : neg crtl) mm/dd/yy
1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng new BioBrick #:1 (Pick #)
2. vector(c/d)/ ## ng  
  1 2
Insert DNA ### ---
Vector DNA ### ###
2x lgn buf (Roche) ### ###
T4 ligase (NEB) 1.0 1.0
dH2O ### ###
  # μL # μL

Oligo annealing

  1. New BB 1
  2. New BB 2
DNA (oligos, 100 μM) up to 18 μL (3 μL ea.)
10x annealing buffer 2.0
dH2O ---
  20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight



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