Search found 14 matches

by roxanalibe
Thu Aug 19, 2010 9:37 am
Forum: Molecular Biology
Topic: cDNA synthesis
Replies: 2
Views: 1963

recently I used the same protocol (first strand dna synthesis superscript from invitrogen, digest the leftovers of RNA by RNaseH from the kit) and it worked fine for me, because I used 3 times the amounts for the reactions and put 12 ug RNA (i extracted by quiagen kit for total RNA and had a final c...
by roxanalibe
Mon Aug 09, 2010 9:52 am
Forum: Cell Biology
Topic: decomplemeted serum
Replies: 0
Views: 1213

decomplemeted serum

Hi everybody, I need to grow my cells in serum without any cytokine that comes in the serum. So, I need to eliminate the traces of cytokines from fetal bovine serum OR I need to find a commercial serum that has no cytokines. How can I do that? One idea could be that serum inactivation at 56°C for 30...
by roxanalibe
Mon Jul 26, 2010 2:21 pm
Forum: Molecular Biology
Topic: pCMV-SPORT6 empty vector
Replies: 4
Views: 3558

in this way i will never learn how to do it :-)
by roxanalibe
Mon Jul 26, 2010 9:17 am
Forum: Molecular Biology
Topic: pCMV-SPORT6 empty vector
Replies: 4
Views: 3558

pCMV-SPORT6 empty vector

Hi, I need to obtain the empty vector pCMV-SPORT6, for control, and I never obtained an empty vector out of an one with an insert. Any idea how I could do that (tips for a better ligation of the empty vector)? the mcs has: ecorI, xmaI, smaI, ecorV, salI, notI,xbaI, xhoI...the insert is cloned with s...
by roxanalibe
Mon Jul 26, 2010 9:13 am
Forum: Molecular Biology
Topic: PCR product of mutagenesis_good but still wrong
Replies: 4
Views: 2781

it might be something wrong, as there is no way I can get a normal pattern for none of the miniprep I did from the transformation plate with the initial positive clone. the primers for seq are 100 bp distance from the mutation....still, I think I need to repeat the mutagenesis...and I begin to get s...
by roxanalibe
Tue Jul 20, 2010 5:15 pm
Forum: Molecular Biology
Topic: PCR product of mutagenesis_good but still wrong
Replies: 4
Views: 2781

ok, my boss unlighted me, and hope is this the answer to my problem: the miniprep quality could be affected in the purification process and by seq the DNA it can appear a duplication but on different frames (I am always using 3 frames way to read the DNA sequencing), and what can appear to be a dupl...
by roxanalibe
Tue Jul 20, 2010 1:44 pm
Forum: Molecular Biology
Topic: PCR product of mutagenesis_good but still wrong
Replies: 4
Views: 2781

PCR product of mutagenesis_good but still wrong

Hallo, I have this problem introducing a point mutation in an insert, with primers recommended by PrimerX_ Primer Design Based on DNA Sequence. I know the primers are good, as I introduced the same mutation in the same insert but different vector. I cannot cut it from there and put it in the vector ...
by roxanalibe
Thu Jul 15, 2010 10:12 am
Forum: Molecular Biology
Topic: Antibody works in Immunofluorescence but not in Western blot
Replies: 2
Views: 3181

if it helps, i asked around and apparently we really have in the lab antiboodies that recognize the 3D structure of the protein, so the epitope is not linear, it is impossible to use them for denaturated protein.
by roxanalibe
Thu Jul 15, 2010 10:02 am
Forum: Molecular Biology
Topic: enzyme digestion of a PCR product
Replies: 3
Views: 1877

thank you, I already used the double-digestion NEB site, but I was thinking that in the case of just a PCR product is different from a plasmid, for which I usually dont have any problem to use double-digestion.
by roxanalibe
Thu Jul 15, 2010 9:07 am
Forum: Molecular Biology
Topic: enzyme digestion of a PCR product
Replies: 3
Views: 1877

enzyme digestion of a PCR product

Hello, I need to cut with EcoR I and XhoI a PCR product in which I introduced the restriction sites for these enzyme and I am wondering if I should cut with both of them in the same time and purify from the gel, and then perform ligation, or I should cut with one at a time and then purify after each...
by roxanalibe
Wed Jul 14, 2010 7:53 am
Forum: Molecular Biology
Topic: Latrunculin A and cytokine treatment in the same time
Replies: 2
Views: 2702

thanks a lot, this is useful and fast, as i will begin the treatment in one hour
by roxanalibe
Tue Jul 13, 2010 4:54 pm
Forum: Molecular Biology
Topic: Antibody works in Immunofluorescence but not in Western blot
Replies: 2
Views: 3181

sometimes the antibodies made against the native proteins, and easy to use for IF or FACS could be bad to recognize the same protein in denaturating condition (WB); moreover there are antibodies that work very well in FACS but bind non-specific to many denaturated proteins, so they cannot be used in...