Haynes Lab:Notebook/Synthetic Chromatin for Cancer Research/2015/06/24: Difference between revisions
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(Autocreate 2015/06/24 Entry for Haynes_Lab:Notebook/Synthetic_Chromatin_for_Cancer_Research) |
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== | ==6-24-15== | ||
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---- | |||
Assemblies: linkers/hPCD construct & PcTF (KAH 126) | |||
* Assemblies | |||
# DT013_V0120: DT001(E/S)/252 + PcTF_V0120(E/X)/5080 | |||
# DT014_V0120: DT002(E/S)/432 + PcTF_V0120(E/X)/5080 | |||
# DT015_V0120: DT003(E/S)/252 + PcTF_V0120(E/X)/5080 | |||
# DT017_V0120: DT004(E/S)/432 + PcTF_V0120(E/X)/5080 | |||
<!-- ##### | |||
* Digests (Fermentas FD) | |||
{| {{table}} cellspacing="3" <!-- Digest rxn. table --> | |||
|- valign="top" | |||
| bgcolor=#cfcfcf | Reagent | |||
| bgcolor=#cfcfcf | Rxn1 | |||
| rowspan="7" | Expected bands:<br>1. fshPCD(E/S) = 3200, 186*<br><br>(*) Bands that were cut out for purification | |||
| rowspan="7" | [[Image:6.24.15.jpg|250px|]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | |||
** Well 1 was punctured so the ladder was put into Well 3 as well for safe measures. | |||
|- | |||
| DNA (plasmid) || 25.0 | |||
|- | |||
| 10x buffer || 3.0 | |||
|- | |||
| EcoRI || 1.0 | |||
|- | |||
| enzyme 2 || 1.0 | |||
|- | |||
| dH<sub>2</sub>O || --- | |||
|- | |||
| || 30 μL | |||
|} | |||
--> 37°C/ ~30 min. | |||
* Gel purification | |||
** Sigma GenElute Gel purification kit | |||
** Elute & back-elute w/ 25 μL elution sln. | |||
* Measure conc.'s | |||
{| {{table}} cellspacing="3" <!-- [DNA] table --> | |||
|- bgcolor=#cfcfcf | |||
| Sample || OD260 || 260/280 || ng/μL | |||
|- | |||
| 1. DT001(E/S) || --- || --- || --- | |||
|- | |||
| 2. DT002(E/S) || --- || --- || --- | |||
|- | |||
| 3. DT003(E/S) || --- || --- || --- | |||
|- | |||
| 4. DT004(E/S) || --- || --- || --- | |||
|- | |||
| 5. PcTF_V0120(E/X) || --- || --- || --- | |||
|} | |||
** Readings were odd, which is typical for the Sigma gel extraction products. | |||
** Will use an arbitrary volume of 2.0 insert and 2.0 vector as it has worked before. | |||
** Good insert : vector ratio should not be difficult to achieve since insert is very short. | |||
* Ligations | |||
# DT013_V0120: DT001(E/S)/252 + PcTF_V0120(E/X)/5080 | |||
# DT014_V0120: DT002(E/S)/432 + PcTF_V0120(E/X)/5080 | |||
# DT015_V0120: DT003(E/S)/252 + PcTF_V0120(E/X)/5080 | |||
# DT017_V0120: DT004(E/S)/432 + PcTF_V0120(E/X)/5080 | |||
# PcTF_V0120(E/X)/5080 | |||
* Note: Used one negative (one vector only) | |||
{| {{table}} cellspacing="3" <!-- Ligation rxn table --> | |||
|- valign="top" | |||
| bgcolor=#cfcfcf | Reagent | |||
| bgcolor=#cfcfcf | Rxn1-4 | |||
| bgcolor=#cfcfcf | Rxn5 | |||
|- | |||
| 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: ~200 colonies on the ligation plates, 1 colony on the neg. ctrl plate. | |||
* Picked two colonies from each plate (1-4) for streak library and 5 mL cultures | |||
__NOTOC__ | __NOTOC__ |
Revision as of 18:04, 24 June 2015
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6-24-15Assemblies: linkers/hPCD construct & PcTF (KAH 126)
--> 37°C/ ~30 min.
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