Holmesbr/plan: Difference between revisions
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=Introduction= | =Introduction= | ||
This page is meant to keep my two mentors up-to-date on what I am working on and also help keep myself organized. | This page is meant to keep my two mentors up-to-date on what I am working on and also help keep myself organized. | ||
Anyone can post if they have helpful suggestions. | |||
=Specific Aims= | =Specific Aims= | ||
==RNAi Knockdown of RNase H1 <i>in situ</i>== | ==RNAi Knockdown of RNase H1 <i>in situ</i>== | ||
===What I have done=== | |||
*miRNA | *miRNA | ||
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*Analysis of RNase H1 Knock-down by RNase H1 Activity Gel (aka Renaturation Assay) | *Analysis of RNase H1 Knock-down by RNase H1 Activity Gel (aka Renaturation Assay) | ||
I harvested a known quanity of Hos and 3T3 cells 5 days previous, pelleted and froze at -20. I ran a seriel dilution of these sample as well as 7.6 and .76 ng of RNase H1 as a positive control. I incubated one night, and showed 0 RNase H1 activity, even in the positive control. I incubated a second night. Still no RNase H1 activity. | I harvested a known quanity of Hos and 3T3 cells 5 days previous, pelleted and froze at -20. I ran a seriel dilution of these sample as well as 7.6 and .76 ng of RNase H1 as a positive control. I incubated one night, and showed 0 RNase H1 activity, even in the positive control. I incubated a second night. Still no RNase H1 activity. | ||
===What Next?=== | |||
*Test miRNA on Exogenous RNase H1 by co-transfecting with M27-RNase H1-GFP | |||
*Using the lower level of cell number, try to opimize TE and also harvest RNA to make sure enough RNA can be harvested. | |||
*Re-do the activity gel. It should have worked. Maybe use FRESH 3T3 and Hos samples. | |||
==Immunolabeling RNase H1, Top3a and POLG== | ==Immunolabeling RNase H1, Top3a and POLG== |
Revision as of 13:04, 14 June 2006
Introduction
This page is meant to keep my two mentors up-to-date on what I am working on and also help keep myself organized.
Anyone can post if they have helpful suggestions.
Specific Aims
RNAi Knockdown of RNase H1 in situ
What I have done
- miRNA
I have 4 miRNA constructs plus a negative control miRNA construct. I have tested these against endogenous RNase H1 in 3T3 cells, but the knock-down by RT-RT-qPCR has been abysmal, with only one construct acheiving ~40% knock-down. However, this may be due to poor Transfection Efficiency.
- Transfection Efficiency
The latest results (6/14/6) indicate that maybe I am plating 4X too many cells on Day 0 before transfecting (by FACS analysis, I was able to increase the TE from 50%to 80%).
- Analysis of RNase H1 Knock-down by RNase H1 Activity Gel (aka Renaturation Assay)
I harvested a known quanity of Hos and 3T3 cells 5 days previous, pelleted and froze at -20. I ran a seriel dilution of these sample as well as 7.6 and .76 ng of RNase H1 as a positive control. I incubated one night, and showed 0 RNase H1 activity, even in the positive control. I incubated a second night. Still no RNase H1 activity.
What Next?
- Test miRNA on Exogenous RNase H1 by co-transfecting with M27-RNase H1-GFP
- Using the lower level of cell number, try to opimize TE and also harvest RNA to make sure enough RNA can be harvested.
- Re-do the activity gel. It should have worked. Maybe use FRESH 3T3 and Hos samples.