Stanford/BIOE44:Module 3:Day1: Difference between revisions
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(New page: {{Template:Stanford/BioE44}} <div style="padding: 5px; width: 656px; border: 2px solid #397D02;"> =M3: Day 1 - What to measure?= ==Introduction== ==In Class== ===What happened with our gel...) |
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==In Class== | ==In Class== | ||
===What happened with our gel extractions?=== | ===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. | |||
====The Results==== | |||
[[Image:100426-gelextraction.tif|center|Gel extraction test.]] | |||
'''Legend''' | |||
{| style="width:95%" border=1px | |||
|'''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. | |||
|50 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 | |||
|- | |||
|} |
Revision as of 20:33, 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.
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. | 50 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 |