Haynes Lab:Notebook/Synthetic Chromatin for Cancer Research/2015/06/24: Difference between revisions

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
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|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>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</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]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}}
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==Entry title==
==6-24-15==
* Insert content here...
<!-- Precede finished items with a checkmark &#x2713; -->
----
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


<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
 
 
* 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 || ---
|-
| &nbsp; || 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
|-
| &nbsp;            || 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__

Latest revision as of 01:01, 27 September 2017

Project name Main project page
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6-24-15

Assemblies: linkers/hPCD construct & PcTF (KAH 126)

  • Assemblies
  1. DT013_V0120: DT001(E/S)/252 + PcTF_V0120(E/X)/5080
  2. DT014_V0120: DT002(E/S)/432 + PcTF_V0120(E/X)/5080
  3. DT015_V0120: DT003(E/S)/252 + PcTF_V0120(E/X)/5080
  4. DT017_V0120: DT004(E/S)/432 + PcTF_V0120(E/X)/5080


  • Digests (Fermentas FD)
Reagent Rxn1 Expected bands:
1. fshPCD(E/S) = 3200, 186*

(*) Bands that were cut out for purification 		File:6.24.15.jpg
30 μL/lane, 1% agarose; 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
dH2O ---
  30 μL

--> 37°C/ ~30 min.


  • Gel purification
    • Sigma GenElute Gel purification kit
    • Elute & back-elute w/ 25 μL elution sln.


  • Measure conc.'s
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
  1. DT013_V0120: DT001(E/S)/252 + PcTF_V0120(E/X)/5080
  2. DT014_V0120: DT002(E/S)/432 + PcTF_V0120(E/X)/5080
  3. DT015_V0120: DT003(E/S)/252 + PcTF_V0120(E/X)/5080
  4. DT017_V0120: DT004(E/S)/432 + PcTF_V0120(E/X)/5080
  5. PcTF_V0120(E/X)/5080
  • Note: Used one negative (one vector only)
Reagent Rxn1-4 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
dH2O --- 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
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