Haynes Lab:Notebook/Synthetic Biology and Bioinformatics for Predictable Control of Therapeutic Gene2/2013/01/10: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
Caroline Hom (talk | contribs) (Autocreate 2013/01/10 Entry for Haynes_Lab:Notebook/Synthetic_Biology_and_Bioinformatics_for_Predictable_Control_of_Therapeutic_Gene2) |
(fix raw html notebook nav) |
||
(4 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;"> Project name</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</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. ##### --> | ||
== | ==cDNA== | ||
* | '''Preparation''' | ||
* Work with samples on ice | |||
* Ice: RNase H, SS III RT, and RNase out | |||
* Room temp: Oligo nT, RT Buffer, MgCl2, dNTP mix, water, DTT | |||
'''Sample Reading''' | |||
* Add 2μL of each sample plus blank to the plate reader | |||
# K562 Sample 1: 0.44 (260), 2.043 (260/280), 352.1 (ng/mL) | |||
# K562 Sample 2: 0.385, 2.035, 308.3 | |||
# SK-N-SH Sample 1: 0.054, 1.819, 43.3 | |||
# SK-N-SH Sample 2: 0.201, 2.016, 160.7 | |||
*Need to make 8μL solutions of the lowest concentration for each cell line | |||
# Sample 1: 7.1μL cells + 0.9 H2O = 2.5ug | |||
# Sample 2: 8μL cells = 2.5ng | |||
# Sample 3: 8μL cells = 0.35ug | |||
# Sample 4: 2.2μL cells + H2O = 0.35ng | |||
'''Part 1''' | |||
* Make sure all samples are filled up to 8uL (fill rest with water if not) | |||
* Add 1μL of primer (Oligo dT) | |||
* Add 1μL of dNTP | |||
* Incubate for 5 min @ 65°C | |||
* Ice for 1 min | |||
'''Part 2''' | |||
* Make a MM for 4 rxns: need 2μL RT Buffer, 4μL MgCl2, 2μL DTT, 1μL RNaseOut, and 1μL SS III per rxn | |||
* Add 10μL of MM to each rxn tube | |||
* Mix tubes gently, centrifuge for a few seconds | |||
'''Part 3''' | |||
* RT-PCR Machine, set up as follows: | |||
# Stage 1: 50 min @ 50°C | |||
# Stage 2: 5 min @ 85°C | |||
# Stage 3: Infinity @ 4°C | |||
* Add 1μL: of RNase H | |||
* Incubate @ 37°C for 20 min | |||
* Store at -20°C | |||
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | ||
|} | |} |
Latest revision as of 22:20, 26 September 2017
Project name | Main project page Previous entry Next entry |
cDNAPreparation
Sample Reading
Part 1
Part 2
Part 3
|