User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/28: Difference between revisions
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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"| | |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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| colspan="2"| | | colspan="2"| | ||
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<!-- Precede finished items with a checkmark ✓ --> | <!-- Precede finished items with a checkmark ✓ --> | ||
* David Tze - assemblies: linkers + hPCD | * David Tze - assemblies: linkers + hPCD | ||
* Ben - DBN007 5 mL cultures | |||
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* Assemblies | * Assemblies | ||
# | # DT001_pUC57: hPCD(E/S)/186 + PLflex_pUC57(E/X)/2763 | ||
# | # DT002_pUC57: hPCD(E/S)/186 + PLflex4_pUC57(E/X)/2930 | ||
# | # DT003_pUC57: hPCD(E/S)/186 + PLrigid_pUC57(E/X)/2763 | ||
# | # DT004_pUC57: hPCD(E/S)/186 + PLrigid4_pUC57(E/X)/2931 | ||
* Digests (Fermentas FD) | * Digests (Fermentas FD) | ||
{| {{table}} cellspacing="3" <!-- Digest rxn. table --> | {| {{table}} cellspacing="3" <!-- Digest rxn. table --> | ||
|- valign="top" | |- valign="top" | ||
| bgcolor=#cfcfcf | Reagent | | bgcolor=#cfcfcf | Reagent | ||
| bgcolor=#cfcfcf | | | bgcolor=#cfcfcf | Rxn1 | ||
| rowspan="7" | < | | bgcolor=#cfcfcf | Rxn2-5 | ||
| rowspan="7" | Expected bands:<br>1. hPCD(E/S) = 3200, 186*<br>2. PLflex_pUC57(E/X) = 2763*<br>3. PLflex4_pUC57(E/X) = 2930*<br>4. PLrigid_pUC57(E/X) = 2763*<br>5. PLrigid4_pUC57(E/X) = 2931*<br><br>(*) Bands that were cut out for purification | |||
| rowspan="7" | [[Image:KAH052815_gel1.jpg|250px|Hover name]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | |||
|- | |- | ||
| DNA (plasmid) || | | DNA (plasmid) || 25.0 || 15.0 | ||
|- | |- | ||
| 10x buffer || 3.0 | | 10x buffer || 3.0 || 3.0 | ||
|- | |- | ||
| | | EcoRI || 1.0 || 1.0 | ||
|- | |- | ||
| enzyme 2 || 1.0 | | enzyme 2 || 1.0 || 1.0 | ||
|- | |- | ||
| dH<sub>2</sub>O || --- | | dH<sub>2</sub>O || --- || 10.0 | ||
|- | |- | ||
| || 30 μL | | || 30 μL | ||
|} | |} | ||
--> 37°C/ ~30 min. | |||
* Gel purification | |||
** Sigma GenElute Gel purification kit | |||
** Elute & back-elute w/ 25 μL elution sln. | |||
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| Sample || OD260 || 260/280 || ng/μL | | Sample || OD260 || 260/280 || ng/μL | ||
|- | |- | ||
| 1. | | 1. hPCD(E/S) || --- || --- || --- | ||
|- | |||
| 2. PLflex_pUC57(E/X) || --- || --- || --- | |||
|- | |||
| 3. PLflex4_pUC57(E/X) || --- || --- || --- | |||
|- | |||
| 4. PLrigid_pUC57(E/X) || --- || --- || --- | |||
|- | |- | ||
| | | 5. PLrigid4_pUC57(E/X) || --- || --- || --- | ||
|} | |} | ||
** Readings are very odd, which is typical for the Sigma gel extraction products. | |||
** Will try my luck with these and use an arbitrary volume of 2.0 insert and 2.0 vector. | |||
** Good insert : vector ratio should not be difficult to achieve since insert is very short. | |||
* Ligations | |||
** 2:1 ratio calculation: 186 bp insert / ~2800 bp vector * 2 * 50 = 6.64 ng insert | |||
# DT001_pUC57: hPCD(E/S)/186 + PLflex_pUC57(E/X)/2763 | |||
# DT002_pUC57: hPCD(E/S)/186 + PLflex4_pUC57(E/X)/2930 | |||
# DT003_pUC57: hPCD(E/S)/186 + PLrigid_pUC57(E/X)/2763 | |||
# DT004_pUC57: hPCD(E/S)/186 + PLrigid4_pUC57(E/X)/2931 | |||
# PLflex_pUC57(E/X)/2763 | |||
* Note: not enough plates to do neg. ctrls for all vectors. Used just one of the vectors | |||
{| {{table}} cellspacing="3" <!-- Ligation rxn table --> | |||
{| {{table}} cellspacing="3" <!-- | |- valign="top" | ||
|- | |||
| bgcolor=#cfcfcf | Reagent | | bgcolor=#cfcfcf | Reagent | ||
| bgcolor=#cfcfcf | | | bgcolor=#cfcfcf | Rxn1-4 | ||
| bgcolor=#cfcfcf | Rxn5 | |||
|- | |- | ||
| DNA | | Insert DNA || 2.0 || --- | ||
|- | |- | ||
| | | Vector DNA || 2.0 || 2.0 | ||
|- | |- | ||
| | | 2x lgn buf (Roche) || 5.0 || 5.0 | ||
|- | |- | ||
| | | T4 ligase (NEB) || 1.0 || 1.0 | ||
|- | |- | ||
| || | | dH<sub>2</sub>O || --- || 2.0 | ||
|- | |||
| || 10.0 μL || 10.0 μL | |||
|} | |} | ||
* | RESULTS | ||
* Looks successful - David Tze: ~200 colonies on the ligation plates, 1 colony on the neg. ctrl plate. | |||
* David Tze: picked two colonies from each plate (1-4) for streak library and 5 mL cultures | |||
---- | |||
'''Ben - DBN007 5 mL cultures''' | |||
* DBN001_psB1A3: https://benchling.com/s/S2BS6ziD/edit | |||
* DBN006: https://benchling.com/s/XoQEXLOz/edit | |||
* Minipreps<br> | |||
** Sigma GenElute kit, elute w/ 75 μL elution sln. | |||
* Digests | |||
** Cut w/ E/S | |||
{| {{table}} cellspacing="3" <!-- | {| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table --> | ||
|- valign="top" | |- valign="top" | ||
| bgcolor=#cfcfcf | Reagent | | bgcolor=#cfcfcf | Reagent | ||
| bgcolor=#cfcfcf | | | bgcolor=#cfcfcf | Volume | ||
| | | rowspan="7" | <u>Expected:</u><br>1-2. DBN007_pSB1A3 = 2197, 2132<br>DBN001 = 2132, 224<br> | ||
| rowspan="7" | [[Image:KAH052815_gel2.jpg|150px|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 | ||
|- | |- | ||
| | | SpeI || 1.0 | ||
|- | |- | ||
| dH<sub>2</sub>O | | dH<sub>2</sub>O || 9.5 | ||
|- | |- | ||
| | | || 15 μL | ||
|} | |} | ||
--> 37°C/ ~15 min. | |||
CONCLUSIONS | |||
* Looks successful. 2197, 2132 bands are hard to resolve at the 2000 kb range. | |||
* Submit the plasmids for sequencing | |||
Latest revision as of 00:59, 27 September 2017
Karmella's BioBrick Cloning | Main project page Previous entry Next entry | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
05/28/15
David Tze - assemblies: linkers + hPCD
--> 37°C/ ~30 min.
Ben - DBN007 5 mL cultures
--> 37°C/ ~15 min.
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