User:Karmella Haynes/Notebook/BioBrick cloning/2013/03/20: Difference between revisions
From OpenWetWare
(Autocreate 2013/03/20 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning) |
(fix raw html notebook nav) |
||
(19 intermediate revisions by one other user not shown) | |||
Line 2: | Line 2: | ||
|- | |- | ||
|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"| | |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}}}}]]}} | ||
|- | |- | ||
| 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. ##### --> | ||
== | ==03/20/13== | ||
<!-- Precede finished items with a checkmark ✓ --> | <!-- Precede finished items with a checkmark ✓ --> | ||
* | * Oligo annealing, round 2 | ||
* | * Assembly: annealed part into MV2 | ||
---- | ---- | ||
''' | '''Assemblies''' | ||
* | * New strategy for mammalian vector: | ||
** Insert K-XbaI-NLS-6his-stop into the puro vector that has a deleted promoter (MV2, puro). This way, the vector can be customized to carry different promoters. | |||
** Insertion of the ds oligo should destroy the vector's XbaI site | |||
** Will need to check inserts for copy number and orientation | |||
* '''Oligo annealing''' | |||
''' | * See [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2013/03/19 03/19/13] for details about the oligos and reaction set-up | ||
* Redo oligo annealing with 99.9 and 97.9°C starting temperatures... | |||
* Previous Thermal cycling (3/19/13) | |||
** 99.9, 10 min. | |||
** [99.9, 1 min., -2°C; 90.0, 1 min., -2°C] x35 | |||
** 25°C, ∞ | |||
** Labeled tubes A1, A2; Stored on bench o/n | |||
* New Thermal cycling (3/20/13) | |||
** 99.9, 10 min. | |||
** [99.9, 1 min., -2°C; 97.9, 1 min., -2°C] x36 | |||
** 25°C, ∞ | |||
** Labeled tubes A3, A4 | |||
* | * '''Digest''' (Fermentas FD) | ||
** | ** MV2, XbaI only | ||
{| {{table}} cellspacing="3" <!-- Digest rxn. table --> | {| {{table}} cellspacing="3" <!-- Digest rxn. table --> | ||
Line 49: | Line 46: | ||
| bgcolor=#cfcfcf | Reagent | | bgcolor=#cfcfcf | Reagent | ||
| bgcolor=#cfcfcf | Volume | | bgcolor=#cfcfcf | Volume | ||
| rowspan="7" | | | rowspan="7" | [[Image:KAH032013_gel1.jpg|100px|Hover name]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | ||
|- | |- | ||
| DNA (plasmid) || | | DNA (plasmid) || 20.0 μL | ||
|- | |- | ||
| 10x buffer || 3.0 | | 10x buffer || 3.0 | ||
|- | |- | ||
| | | XbaI || 2.0 | ||
|- | |- | ||
| dH<sub>2</sub>O || | | dH<sub>2</sub>O || 5.0 | ||
|- | |- | ||
| || 30 μL --> 37°C/ ~ | | || 30 μL --> 37°C/ ~15 min. | ||
|} | |} | ||
* Gel purify using the Zymo DNA Gel Recovery kit. | |||
* Elute with 20 μL dH<sub>2</sub>O | |||
* Measure conc.' | |||
* '''Measure conc.''' | |||
{| {{table}} cellspacing="3" <!-- [DNA] table --> | {| {{table}} cellspacing="3" <!-- [DNA] table --> | ||
|- bgcolor=#cfcfcf | |- bgcolor=#cfcfcf | ||
| Sample || OD260 || 260/280 || ng/μL | | Sample || OD260 || 260/280 || ng/μL | ||
|- | |- | ||
| 1. | | 1. MV2 (XbaI) || 0.081 || 1.841 || 81.3 | ||
| | |||
| | |||
|} | |} | ||
* Dephosphorylation (Roche) | * '''Dephosphorylation''' (Roche) | ||
{| {{table}} cellspacing="3" <!-- Dephos table --> | {| {{table}} cellspacing="3" <!-- Dephos table --> | ||
|- | |- | ||
Line 82: | Line 79: | ||
| bgcolor=#cfcfcf | Volume | | bgcolor=#cfcfcf | Volume | ||
|- | |- | ||
| | | MV2 (XbaI) || 6.2 μL (500 ng) | ||
|- | |- | ||
| 10x buffer d.p. || 2.0 | | 10x buffer d.p. || 2.0 | ||
|- | |- | ||
| phosphatase || 1.0 | | rapid alk. phosphatase || 1.0 | ||
|- | |- | ||
| dH<sub>2</sub>O || | | dH<sub>2</sub>O || 10.8 | ||
|- | |- | ||
| || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL | | || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL | ||
Line 94: | Line 91: | ||
* Ligations | * '''Ligations''' | ||
{| {{table}} cellspacing="3" <!-- Ligations table --> | {| {{table}} cellspacing="3" <!-- Ligations table --> | ||
|- bgcolor=#cfcfcf | |- bgcolor=#cfcfcf | ||
| Ligation || <font color="blue"><u>Plate results (lig : neg crtl)</u> | | Ligation || <font color="blue"><u>Plate results (lig : neg crtl)</u> 03/21/13</font> | ||
|- | |- | ||
| 1. | | 1. A1 (ds oligo S/S)/76, ''0.5 μL'' + MV2(X dp)/4529, 25 ng || <font color="red">MV9 failed</font> | ||
|- | |- | ||
| 2. | | 2. A1 (ds oligo S/S)/76, ''1.0 μL'' + MV2(X dp)/4529, 25 ng || <font color="red">MV9 failed</font> | ||
|- | |||
| 3. A1 (ds oligo S/S)/76, ''2.0 μL'' + MV2(X dp)/4529, 25 ng || <font color="red">MV9 failed</font> | |||
|- | |||
| 4. MV9(X dp)/ 25 ng || | |||
|- | |||
| 5. A3 (ds oligo S/S)/76, ''0.5 μL'' + MV2(X dp)/4529, 25 ng || <font color="red">MV9 failed</font> | |||
|- | |||
| 6. A3 (ds oligo S/S)/76, ''1.0 μL'' + MV2(X dp)/4529, 25 ng || <font color="red">MV9 failed</font> | |||
|- | |||
| 7. A3 (ds oligo S/S)/76, ''2.0 μL'' + MV2(X dp)/4529, 25 ng || <font color="red">MV9 failed</font> | |||
|- | |||
| 8. MV9(X dp)/ 25 ng || | |||
|- | |||
| 9. MV9(X no dp)/ 25 ng + T4 ligase || <font color="blue">Re-ligation successful, ~40 colonies</font> | |||
|- | |||
| 10. MV9(X no dp)/ 25 ng || | |||
|} | |} | ||
{| {{table}} cellspacing="3" <!-- Ligation rxn table --> | {| {{table}} cellspacing="3" <!-- Ligation rxn table --> | ||
| || 1 || 2 || | | || 1 || 2 || 3 || 4 || 5 || 6 || 7 || 8 || 9 || 10 | ||
|- | |- | ||
| Insert DNA || | | Insert DNA || 0.5 || 1.0 || 2.0 || --- || 0.5 || 1.0 || 2.0 || --- || --- || --- | ||
|- | |- | ||
| Vector DNA || | | Vector DNA || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 || 0.3 || 0.3 | ||
|- | |- | ||
| 2x lgn buf (Roche) || | | 2x lgn buf (Roche) || 5.0 || 5.0 || 5.0 || 5.0 || 5.0 || 5.0 || 5.0 || 5.0 || 5.0 || 5.0 | ||
|- | |- | ||
| T4 ligase (NEB) || 1.0 || 1.0 || | | 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 || | | dH<sub>2</sub>O || 2.5 || 2.0 || 1.0 || 3.0 || 2.5 || 2.0 || 1.0 || 3.0 || 3.7 || 4.7 | ||
|- | |- | ||
| || | | || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL | ||
|} | |} | ||
* 10 min./ room temp | |||
* Add 30 μL DH5α; 5 min/ ice | |||
* Plate on 100 μg/mL amp; incubate at 37°C | |||
---- | ---- | ||
3/21/13<br> | |||
Results for plates 9 and 10 | |||
| | [[Image:KAH032113_plates1.jpg|300px|Hover name]] | ||
| | |||
Latest revision as of 22:33, 26 September 2017
Karmella's BioBrick Cloning | Main project page Previous entry Next entry | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
03/20/13
Assemblies
3/21/13 Results for plates 9 and 10
|