Stanford/BIOE44:Module 3:Day1: Difference between revisions
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#For K274003 (this is what I used to test the gel extractions) I separated the digestion into 3 wells on a gel. The other digests each got their own well. (0.8% agarose) | #For K274003 (this is what I used to test the gel extractions) I separated the digestion into 3 wells on a gel. The other digests each got their own well. (0.8% agarose) | ||
#I cut out each band and gel extracted with and without isopropanol for the K274003 pieces. The rest were extracted with a Zymo kit. | #I cut out each band and gel extracted with and without isopropanol for the K274003 pieces. The rest were extracted with a Zymo kit. | ||
#After extraction I ran 0.5ul on a gel to determine concentration. | #After extraction I ran 0.5ul on a gel to determine concentration (this is the gel in the image below). | ||
====The Results==== | ====The Results==== | ||
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===Useful tools=== | |||
'''Literature''' | |||
*[http://www.ncbi.nlm.nih.gov/pubmed/ PubMed] | |||
'''Sequence Viewing and Manipulation''' | |||
*[http://www.biology.utah.edu/jorgensen/wayned/ape/ ApE] (free download) |
Revision as of 20:42, 26 April 2010
M3: Day 1 - What to measure?
Introduction
In Class
What happened with our gel extractions?
What did Isis do?
I had a hunch that the isopropanol step was messing up our gel extractions so I decided to test this. I also prepared some color generators and vectors.
- I digested 10ul K274003, K274100, K274200 with XbaI/PstI and K274220 (pSB2k3 vector) with EcoRI/PstI. This is roughly 2ug of DNA for each.
- For K274003 (this is what I used to test the gel extractions) I separated the digestion into 3 wells on a gel. The other digests each got their own well. (0.8% agarose)
- I cut out each band and gel extracted with and without isopropanol for the K274003 pieces. The rest were extracted with a Zymo kit.
- After extraction I ran 0.5ul on a gel to determine concentration (this is the gel in the image below).
The Results
Legend
Label | Meaning | Concentration |
1kb ladder | NEB 1kb ladder | |
003 with IP | Part K274003 (green generator) cut at XbaI/PstI and gel extracted using Qiagen kit with isopropanol step included. | 50 ng/ul |
003 without IP | Part K274003 (green generator) cut at XbaI/PstI and gel extracted using Qiagen kit with no isopropanol step. | 50 ng/ul |
003 Zymo | Part K274003 (green generator) cut at XbaI/PstI and gel extracted using Zymo kit. | ~75 ng/ul |
100 XbaI/PstI | Part K274100 (red generator) cut at XbaI/PstI, gel extracted with Zymo kit | 250 ng/ul |
200 XbaI/PstI | Part K274200 (orange generator) cut at XbaI/PstI, gel extracted with Zymo kit | 250 ng/ul |
pSB2K3 EcoRI/PstI | Vector pSB2K3 cut at EcoRI/PstI, gel extracted with Zymo kit | 150 ng/ul |
Useful tools
Literature
Sequence Viewing and Manipulation
- ApE (free download)