Stanford/BIOE44:Module 3:Day1

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.

1. I digested 10ul K274003, K274100, K274200 with XbaI/PstI and K274220 (pSB2k3 vector) with EcoRI/PstI. This is roughly 2ug of DNA for each.
2. 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)
3. I cut out each band and gel extracted with and without isopropanol for the K274003 pieces. The rest were extracted with a Zymo kit.
4. After extraction I ran 0.5ul on a gel to determine concentration.

The Results

Gel extraction test.

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