User:Anthony Salvagno/Notebook/Research/2009/04/03/Digestion of pBS
The other day, Kelly managed to scrounge up some pBluescript. We prepped the plasmid and inserted them into E. coli cells. Yesterday Kelly inoculated the E. coli cells and today I gave them a good miniprepping. Then I had my way with them.
Miniprep
I followed the protocol for the Qiagen columns. I asked a simple question on what the best way to spin down the cells would be and Kelly and Toyoko got into an argument about it. It wasn't very heated, but I felt bad nonetheless. Toyoko ended up being right though. After the miniprep, I got a nanodrop reading:
- 156.8ng/uL
or
- 15.68ug (in 100uL)
Bummer. Looks like something didn't go right. We are going to run the process again next week to get more pBS. Can't have too much plasmid.
- Steve Koch 22:12, 3 April 2009 (EDT): Are these the normal sized Qiagen columns? I never did miniprep with Qiagen. But the normal qiagen columns have a max binding capacity of about 10 ug of DNA. I checked their miniprep manual, and your yield seems about right: http://www1.qiagen.com/HB/QIAprepMiniprepKit_EN
Anthony Salvagno 23:53, 5 April 2009 (EDT):I just read the manual you provided. Looks like you/they are right. The amount we got is normal. I should tell Kelly. After I told him how much we got he said, "I guess we should have split it up into 6 tubes."
- Steve Koch 00:07, 6 April 2009 (EDT): Well, if he was arguing to use 6 columns to get more DNA, then he could be correct. Just depends how much DNA you were loading onto them. Usually, for me, though, a fully-loaded column is enough starting material, and I'd rather have high concentration, versus more total mass, but diluted into more elutions.
Digestions
| Tube Number | Vol pBS DNA | Vol/# Buffer 10x | Vol BSA 10x | Vol XhoI | Vol EcoRI | Vol H2O | Total Vol |
|---|---|---|---|---|---|---|---|
| 1 | 32uL | 2/5uL | 5uL | 4uL | -- | 4uL | 50uL |
| 2 | 32uL | Eco Buff/5uL | 5uL | 2uL | 2uL | 4uL | 50uL |
After about 1 hour add 1uL CIP to tube 1. Let reaction run another 30 minutes.
Gel
AAAAAAAAAAAAAaaaaaaaaaHHHHHHHHHHHHHhhhhhhhhhhhh!
Gel was run ok. I'm not gonna post a picture of this one.
I cut out the bands from the gel and on Monday will do the gel extraction kit and probably the ligation reaction of all my stuff. Lets hope this works!
