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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: #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> </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]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} |
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| <!-- ##### 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. ##### --> |
| ==mm/dd/yy== | | ==12/23/14== |
| <!-- Precede finished items with a checkmark ✓ --> | | <!-- Precede finished items with a checkmark ✓ --> |
| * Line item 1 | | * Planning: Gal4 fusion cloning |
| * Line item 2
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| ----
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| '''Minipreps'''<br>
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| * Check with E/P digests
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| {| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
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| |- valign="top"
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| | 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>
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| | 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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| | 10X buffer || 1.5
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| | EcoRI || 1.0
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| | PstI || 1.0
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| | dH<sub>2</sub>O || 9.5
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| | || 15 μL --> 37°C/ ~15 min.
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| |}
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| ---- | | ---- |
| '''Assemblies''' | | '''Planning: Gal4 fusion cloning'''<br> |
| # BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
| | * "Backbone" = Gal4-mCherry-VP64 construct KAH60/pcVN |
| # BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
| | * Approach: swap-in activator domain to replace VP64 |
| | | ** PCR-amplify backbone (omitting VP64) |
| | | ** PCR-amplify insert |
| * Digests (Fermentas FD)
| | ** Plan A: use LCR to ligate insert into backbone |
| ** Specific notes
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| {| {{table}} cellspacing="3" <!-- Digest rxn. table -->
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| |- valign="top"
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| | 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] -->
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| | DNA (plasmid) || up to 25 μL
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| | 10x buffer || 3.0
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| | enzyme 1 || 1.0
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| | enzyme 2 || 1.0
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| | dH<sub>2</sub>O || ---
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| | || 30 μL --> 37°C/ ~30 min.
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| |}
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| * Measure conc.'s | |
| {| {{table}} cellspacing="3" <!-- [DNA] table -->
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| |- bgcolor=#cfcfcf
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| | Sample || OD260 || 260/280 || ng/μL
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| | 1. Digested part (a/b) || --- || --- || ---
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| | 2. Digested part (c/d) || --- || --- || ---
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| |}
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| * Dephosphorylation (Roche)
| | '''Order Primers''' (01/02/15)<br> |
| {| {{table}} cellspacing="3" <!-- Dephos table -->
| | Attempt 1: swap-in ATF activation domain; add 5' phosphates to all oligos |
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| | * KAH60 "backbone" (without VP64, includes scars) = 6637 |
| | bgcolor=#cfcfcf | Reagent
| | ** KAH60 F1 |
| | bgcolor=#cfcfcf | Volume
| | ** KAH60 R1 |
| |-
| | * ATF2 insert = 906 |
| | DNA (clean digest) || up to 17 μL (500 ng)
| | ** ATF2_f1 |
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| | ** ATF2_r1 |
| | 10x buffer d.p. || 2.0
| | * LCR Bridging oligos |
| |-
| | ** LCRb_KAH60_ATF2_1 |
| | phosphatase || 1.0
| | ** LCRb_KAH60_ATF2_2 |
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| | dH<sub>2</sub>O || ---
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| |-
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| | || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
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| |}
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| * Ligations
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| {| {{table}} cellspacing="3" <!-- Ligations table -->
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| |- bgcolor=#cfcfcf
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| | Ligation || <font color="blue"><u>Plate results (lig : neg crtl)</u> mm/dd/yy</font>
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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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| | 2. vector(c/d)/ ## ng ||
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| |}
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| {| {{table}} cellspacing="3" <!-- Ligation rxn table -->
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| | || 1 || 2 ||
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| | Insert DNA || ### || --- ||
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| | Vector DNA || ### || ### ||
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| | 2x lgn buf (Roche) || ### || ### ||
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| | T4 ligase (NEB) || 1.0 || 1.0 ||
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| | dH<sub>2</sub>O || ### || ### ||
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| | || # μL || # μL ||
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| |}
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| ----
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| '''Oligo annealing'''
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| # New BB 1
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| # New BB 2
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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 (3 μL ea.)
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| | 10x annealing buffer || 2.0
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| | dH<sub>2</sub>O || ---
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| | || 20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight
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| |}
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Karmella's BioBrick Cloning
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