Search found 45 matches

by bravebeaker
Thu Aug 29, 2013 7:14 am
Forum: Molecular Biology
Topic: Gel Electrophoresis Help!
Replies: 3
Views: 3100

size of your vector/insert.
gel is prestained with ethidium bromide or not.
what are the restriction enzymes you are using?
did you check if your vector contains multiple sites of the enzymes you are using?

The bands are way up, higher than 10k bp..
by bravebeaker
Mon Aug 26, 2013 5:45 am
Forum: Molecular Biology
Topic: Transcrption factor-promoter (cis-element) interaction
Replies: 3
Views: 3090

How about EMSA then? electromobility shift assay also applicable for the same purpose more or less...
by bravebeaker
Thu Aug 22, 2013 3:31 am
Forum: Molecular Biology
Topic: Transcrption factor-promoter (cis-element) interaction
Replies: 3
Views: 3090

are you testing in plant or animal cells.
I have been working with plant cells and the most common and easy way I guess is the reporter gene assay.
the promoter in question is designed in a vector carrying the GUS reporter gene then you can measure GUS activity for instance...
by bravebeaker
Wed Jul 10, 2013 2:39 am
Forum: Molecular Biology
Topic: Promoter PCR primer set problem
Replies: 2
Views: 3839

Thank you for the help and the link. I am going to give it another try if not I am going to redesign the the Fw primer.
I calculated the Tm by using the 2+4 rule but it seems its not very accurate.
by bravebeaker
Tue Jul 09, 2013 3:05 am
Forum: Molecular Biology
Topic: Promoter PCR primer set problem
Replies: 2
Views: 3839

Promoter PCR primer set problem

Hi all, Recently I have designed the following primers to amplify a part of the promoter region of my gene of interest. fw: 5'- gggg CCGCGG TG AGC AAG TAT ACC AAC CAT -3' (Sacii) Tm 63.3 rv: 5'- gggg TCTAGA CAA TGT ACA CCA ACA TAT AC -3' (Xbai) Tm 56.5 I am using genomic DNA as template and KOD kit....
by bravebeaker
Thu May 02, 2013 5:36 am
Forum: Molecular Biology
Topic: Problem with the normalization of PCR Ct values
Replies: 5
Views: 11058

Jackbean I agree with you, quantification of the pooled cDNA for the standard curve will result in knowing the conc. of the gene of interest.

Tzua, any paper dealing with the expression level of a certain gene using RT-qPCR would be suffice I guess.
by bravebeaker
Wed May 01, 2013 1:12 am
Forum: Molecular Biology
Topic: Problem with the normalization of PCR Ct values
Replies: 5
Views: 11058

in my case when I have a gene of interest and the standard gene (usually actin) I divide the calculated conc. in fmol of the gene of interest by the calculated conc. of actin. about the ct values, the higher the value, less copies are there, if you make a standard curve you can see how the ct values...
by bravebeaker
Mon Apr 22, 2013 9:39 am
Forum: General Discussion
Topic: Boiling alcohol
Replies: 0
Views: 1120

Boiling alcohol

Hi all, I was wondering about boiling alcohol and its effect on how much alcohol retains after it, I read a couple of articles on the internet and found out to completely abolish alcohol you need to boil for 3 hours. Now I am addressing the academics here does anyone have a scientific proof for that...
by bravebeaker
Wed Apr 17, 2013 2:36 am
Forum: Molecular Biology
Topic: Nuclear protein purification
Replies: 4
Views: 3331

Now I totally understand it! Thank you very much.
by bravebeaker
Mon Apr 15, 2013 6:58 am
Forum: Molecular Biology
Topic: Nuclear protein purification
Replies: 4
Views: 3331

Thank you for the elaboration, I know its been almost a month for me to reply I am sorry for that but I finally tried the experiment and the volume I am adding to is 5 ml. But I am not sure how we reached the 2/3 ratio of the original volume. because 2/3 = .66 and if we multiply (.66)(5ml) I will ge...
by bravebeaker
Mon Mar 18, 2013 7:49 am
Forum: Molecular Biology
Topic: Nuclear protein purification
Replies: 4
Views: 3331

Nuclear protein purification

Hi all, During nuclear protein purification I came across the step where I need to add 5M NaCl slowly with shaking until I adjust the molarity to 2M NaCl, how many mls do I need to add? Honestly I tried to tackle the problem in different ways but I couldnt, any help would be really appreciated. Than...
by bravebeaker
Mon Mar 18, 2013 6:20 am
Forum: Molecular Biology
Topic: significant difference
Replies: 2
Views: 2135

I also thought so, thank you!