Search found 37 matches

by snowcapk
Thu Aug 14, 2008 9:48 pm
Forum: Molecular Biology
Topic: Gel extraction kit
Replies: 1
Views: 1461

The main purpose of running the fragment on the gel is to remove impurities, like you said: you can cut out the band (assuming there are multiple bands) that contains the fragment you want and extract it from the gel. You should always run a gel of your PCR product anyway to check for impurities and...
by snowcapk
Tue Aug 12, 2008 4:00 am
Forum: Molecular Biology
Topic: Glycine in Western Blot buffers
Replies: 2
Views: 18159

Are you using discontinuous gels (the kind with stacking and resolving portions)? I have heard an explanation for why glycine is used in the running buffer for those gels. The idea is to keep proteins from separating by size until they reach the resolving portion. To keep the protein in a tight band...
by snowcapk
Thu Aug 07, 2008 5:21 am
Forum: Molecular Biology
Topic: 5 questions without answers...!
Replies: 2
Views: 3421

Re: 5 questions without answers...!

1. A fusion gene for a membrane protein Y is fused to GFP. After transfection, where would I expect to see fluorescent signal first appearing? Is this based on a ribosomal location? Membrane? Membrane of rough ER, assuming you do not disrupt the N-terminal ER signal sequence of the membrane protein...
by snowcapk
Sat Aug 02, 2008 1:59 am
Forum: Molecular Biology
Topic: Porblems making DIG probe - please help
Replies: 5
Views: 6419

Ethidium intercalates into nucleic acids; I don't think it binds to free nucleotides well. This is probably why you cannot see them in an EtBr gel. If you really think that you might not have any nucleotides in there, you could use a spectrophotometer to check the concentration. You need to run a sp...
by snowcapk
Fri Aug 01, 2008 7:36 pm
Forum: Molecular Biology
Topic: Morpholino review: how to knockdown
Replies: 7
Views: 17145

Re: Morpholino review: how to knockdown

Thank you both for your insightful replies! I'm familiar with the rescue experiment you both mentioned (rescue through coinjection with a construct containing the gene's coding region in its native regulatory context, e.g. a BAC) and it is clearly a powerful tool. As you know these constructs tend t...
by snowcapk
Wed Jul 30, 2008 8:30 am
Forum: Molecular Biology
Topic: Morpholino review: how to knockdown
Replies: 7
Views: 17145

Hi Chaka, I'm interested in the different controls you used to check nonspecific effects. What's the standard on this? We do other types of controls (control MASO injection to test for toxicity at a given concentration, in a given batch of embryos; design of reporter constructs to test the efficienc...
by snowcapk
Wed Jul 30, 2008 8:06 am
Forum: Molecular Biology
Topic: Porblems making DIG probe - please help
Replies: 5
Views: 6419

Hi chaka8, It sounds like you've considered everything that I would have - sorry I can't be more helpful! I don't actually know about the incubation temp., but it doesn't seem like it should be a problem. Your cleanup method sounds fine - I use a column from a Qiagen PCR purification kit or Zymo Res...
by snowcapk
Tue Jul 29, 2008 7:47 am
Forum: Molecular Biology
Topic: Porblems making DIG probe - please help
Replies: 5
Views: 6419

A couple of questions: ⋅ Have you tried the controls that come with the kit? If you didn't use the controls, are you sure that you've matched the correct polymerase to the correct restriction digest product? ⋅ Are you sure your DIG "mix" contains the other NTPs? Are you...
by snowcapk
Mon Jul 28, 2008 3:21 pm
Forum: Cell Biology
Topic: Questions for a helpful Cell Biologist
Replies: 3
Views: 2604

Re: Questions for a helpful Cell Biologist

5.Which degrees get you into what jobs? How much do those jobs pay and how hard it is to get those jobs? Unfortunately a BS doesn't qualify you for many positions. You could be a lab technician, which pays decently (I've heard up to $60K) but is astoundingly boring: you repeat the same simple tasks...
by snowcapk
Sun Jul 27, 2008 9:40 pm
Forum: Molecular Biology
Topic: BIG PROBLEM WITH NORTHERN BLOT
Replies: 2
Views: 2222

Your RNA gel probably contained formaldehyde and you probably denatured your samples (including the ladder) with heat and formaldehyde before loading them onto the gel. DNA weight markers are generally double-stranded, but under these conditions the DNA MW marker would be single-stranded just like y...
by snowcapk
Sun Jul 27, 2008 9:27 pm
Forum: Molecular Biology
Topic: Multigene cloning - Looking for a cheap solution
Replies: 1
Views: 1076

Re: Multigene cloning - Looking for a cheap solution

What do you mean by 5 fragments in one vector? Do you want (a) to clone each fragment separately, or do you want (b) to insert all five of them in a row in a single vector? (a) Primers are very cheap. Why don't you just design ten primers with rare restriction sites in the tails, amplify your five f...
by snowcapk
Tue May 13, 2008 8:10 am
Forum: Molecular Biology
Topic: Making SOC solution
Replies: 1
Views: 4272

My suggestions: Make the LB and adjust the pH to 7. I recommend adding Tris at pH 7 to act as a buffer, maintaining the correct pH in the SOC medium as bacteria grow. Autoclave the LB and wait for it to cool. (Mmm, caramel!) Add the glucose, MgCl2, and MgSO4. (I would go for final concentrations of ...