Search found 8 matches

by bluerose
Mon Oct 08, 2007 9:15 pm
Forum: Molecular Biology
Topic: Wouldn't it be possible to use primers with different tm?
Replies: 12
Views: 16282

I am using Taq enzyme and Taq buffer with MgCl2 already in it from Invitrogen.

I can try the other temps, but I'm hoping this 54*C works. I'll let you know how it turns out. Thanks for the tips!
by bluerose
Mon Oct 08, 2007 8:54 pm
Forum: Molecular Biology
Topic: Wouldn't it be possible to use primers with different tm?
Replies: 12
Views: 16282

so ranges of 53*C-55*C are good annealing temperature points? I am trying 54*C right now. It seems 55*C is a common PCR temperature. I may try that next if this run doesn't show anything. I am using this pcr program, going through 30 cycles. I hold at 4*C at the end. 94*C, 1 min 94*C, 30 sec 54*C, 1...
by bluerose
Mon Oct 08, 2007 7:48 pm
Forum: Molecular Biology
Topic: Wouldn't it be possible to use primers with different tm?
Replies: 12
Views: 16282

pcr annealing temperatures

Since this post was already started on the Tm, I'd like to ask a question about the Tm and annealing temperatures for my primers. I have 2 primers that I'm trying to hybridize to genomic DNA. So far I've had no luck in the PCR results. The Tm of my first primer is 61*C and the Tm of my 2nd primer is...
by bluerose
Sat Aug 18, 2007 9:29 pm
Forum: Molecular Biology
Topic: saving my minigel
Replies: 1
Views: 1172

saving my minigel

Is there a way to save my minigel which I ran DNA onto already this morning, so that the bands don't diffuse over the weekend? I was told that if I left it in the TAE buffer, my DNA will diffuse out after a few hours. Is there any way to prevent this long enough and save my gel? I need it for the fu...
by bluerose
Wed Aug 08, 2007 4:17 pm
Forum: Molecular Biology
Topic: question on DNA material?
Replies: 1
Views: 2360

question on DNA material?

I am doing an E. coli plasmid prep on some transformed colonies isolated from agar media and grown up in a liquid culture. I use an alkaline lysis protocol to get the DNA out of the cells, wash with EtOH, then finally resuspend in TE. But before I do resuspend in TE, I see a clean while pellet at th...
by bluerose
Sun Aug 05, 2007 8:36 pm
Forum: Molecular Biology
Topic: plasmid DNA question
Replies: 6
Views: 7313

Well I wish they were. But no, they were not. Even the post doc here gave the same advice, to restreak from the original Agro stock and isolate and purify more plasmid DNA that way. But this wastes a lot of time and you may not get good insert in the end again even if you repeat with same techniques...
by bluerose
Sun Aug 05, 2007 2:36 pm
Forum: Molecular Biology
Topic: plasmid DNA question
Replies: 6
Views: 7313

Thanks for the reply! Apparently, people in the lab I work in don't know this either because they said if I wanted more plasmid, I'd have to go all the way back and isolate the plasmid + insert from Agro cells again! But I have a great purified DNA stock here which I'd like to keep using if I can! I...
by bluerose
Sun Aug 05, 2007 1:57 am
Forum: Molecular Biology
Topic: plasmid DNA question
Replies: 6
Views: 7313

plasmid DNA question

Hello, I have an issue hoping if someone can answer it. I isolated and purified a plasmid with insert from Agrobacterium cells. Following protocol, I injected a small liquid culture of this into E. coli competent cells, heat shocked, and transformed onto agar medium. Transformed colonies grew. I iso...