User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/17: 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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== | ==01/17/13== | ||
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* | * Minipreps: check Golden Gate assembly clones (trial 3) | ||
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'''Minipreps'''<br> | '''Minipreps'''<br> | ||
* Check with E/P digests | * Check with E/P digests | ||
* Minipreps from colonies on Plate #2 (Promega home-made ligation buffer trial) | |||
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table --> | {| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table --> | ||
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| bgcolor=#cfcfcf | Reagent | | bgcolor=#cfcfcf | Reagent | ||
| bgcolor=#cfcfcf | Volume | | bgcolor=#cfcfcf | Volume | ||
| rowspan="7" | <u>Expected:</u><br>1. | | rowspan="7" | <u>Expected:</u><br>1. Plate 2-1 = 2000 (v), 2706 (i)<br>2. Plate 2-2 = 2000 (v), 2706 (i)<br>3. Plate 2-3 (red) = 2000 (v), 2706 (i)<br><br>4. Plate 2-4 = 2000 (v), 2706 (i)<br>5. pSB1A3+RFP = 2000 (v), ~1200 (i)<br>6. hPCD/V0120 = 3200 (v), 186 (i)<br>7. BL01 = 3200 (v), 2520 (i)<br> | ||
| rowspan="7" | | | rowspan="7" | [[Image:KAH011813_gel1.jpg|300px|E/P digests]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | ||
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| DNA(plasmid) || | | DNA(plasmid) || 3.0 μL | ||
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| 10X buffer || 1.5 | | 10X buffer || 1.5 | ||
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Conclusions | |||
''' | * '''Success!''' (1, 2, & 4) Bands match expected size for vector pSB1A3 (not old vector V0120) and for assembled insert: hPCD (186) + BL01 (2520) = 2706 (visibly larger than BL01 band) | ||
* Transferring inserts from V0120 to pSB1A3 is probably a good idea since several internal BsmBI sites might destroy any template background | |||
* Use BsmBI Type IIS method to assemble all the things! | |||
* | |||
* | |||
1/23/13 Sequencing confirmation (DNASU) | |||
# ggPro-1, VF2 - correct sequence, scarless junction between hPCD and BL01 | |||
# ggPro-1, VR - correct sequence for SP1AB of BL01 | |||
# ggPro-2, VF2 - matches ggPro-1 forward (BLAST) | |||
# ggPro-2, VR - matches ggPro-1 reverse (BLAST) | |||
# ggPro-4, VF2 - matches ggPro-1 forward (BLAST) | |||
#ggPro-4, VR - matches ggPro-1 reverse (BLAST) | |||
Latest revision as of 22:22, 26 September 2017
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01/17/13
Minipreps
Conclusions
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