http://www.protocol-online.org/forums Thu, 09 Feb 2012 19:46:36 +0000 360 http://www.protocol-online.org/forums/topic/24360-how-to-prepare-1m-edta-with-only-tris-base/
How do I prepare 1M EDTA, pH 7.4? Only Tris Base is allowed for adjusting the pH, no HCl and NaOH can be added.

Is it even possible to do this? Because I can't seem to find a way.

Thanks for any help.]]> Thu, 09 Feb 2012 19:46:36 +0000 http://www.protocol-online.org/forums/topic/24360-how-to-prepare-1m-edta-with-only-tris-base/ http://www.protocol-online.org/forums/topic/24327-how-to-prepare-digestion-buffer/ This is the list of reagents:


100 mM NaCI
10 mM Tris.CI, pH 8.0
25 mM EDTA, pH 8.0
0.5 % SDS
0.1 mg/ml proteinase

* the final volume should be 1 ml.. can anyone show me the correct steps to prepare each of the materials?

i really appreciate your kindness... Thank you very much.]]> Wed, 08 Feb 2012 07:00:56 +0000 http://www.protocol-online.org/forums/topic/24327-how-to-prepare-digestion-buffer/ http://www.protocol-online.org/forums/topic/24326-trypan-blue-staining/ Is it possible to stain with trypan blue in the petri dish itself, without lifting the cells and counting them in a hemacytometer? If so, does anyone have a protocol for it? Wed, 08 Feb 2012 06:26:36 +0000 http://www.protocol-online.org/forums/topic/24326-trypan-blue-staining/ http://www.protocol-online.org/forums/topic/24303-dna-loading-for-chromatin-check/
I have a relatively basic question. I am starting to do IPs and I have for this to prepare chromatin and qualtiy check it.

After reverese-crosslinking, I am obtaining a descent concentration on the nanodrop i.e: 2371 ng/µl.

after loading 600 ng for the quality check of my sheared chromatin, the bands are very faint although it seems that I have a good shearing. Others show very strong bands using the same amounts !!! The ladder in my case looks fine too.

In order to obtain the 600 ng, I diluted an aliquot of my sample to 1:1 with water so ending with a concentration of 1185 ng/µl from which I took 0.5 µl. To this I added 1.5 µl of Orange G and 8 µl of water to load 10 µl final ...

I am using 1% agarose gel where I load...

Any hint so why I am having a very faint band while 600 ng corresponds to a lot of DNA ?

Many thanks for your suggestions,]]> Mon, 06 Feb 2012 10:48:15 +0000 http://www.protocol-online.org/forums/topic/24303-dna-loading-for-chromatin-check/ http://www.protocol-online.org/forums/topic/24300-control-for-rna-chip-experiment/ Would not adding the crossling reagent (formaldehyde) be a good negative control for an RNA Chip experiment? Sun, 05 Feb 2012 20:58:15 +0000 http://www.protocol-online.org/forums/topic/24300-control-for-rna-chip-experiment/ http://www.protocol-online.org/forums/topic/24296-mes-acetate-buffer-recipe/
I have a protocol which involves the use of "50mM MES-acetate, pH 6.0" buffer. I looked at the MES datasheet and there it is stated that the pKa of MES is 6.15 @ 20C. How can I titrate acetate into MES if the pH of MES is already ~6? Does anyone have a recipe for making MES-acetate pH6?

Thanks!]]> Sun, 05 Feb 2012 13:06:54 +0000 http://www.protocol-online.org/forums/topic/24296-mes-acetate-buffer-recipe/ http://www.protocol-online.org/forums/topic/24284-10-mm-na-acetate-buffer-preparation/
Can anyone guide me on how this 10 mM buffer is prepared or on its calculation.

What currently I do is the old method that we have been following i.,e use 0.123 mg Na Acetate trihydrate + 0.476 µl Gl. Acetic acid, adjust pH to 4.0 using 5 N NaOH. But we do not know how these values have been arrived at and if this calculation is correct.

Please guide me with this regards.

thanks]]> Sat, 04 Feb 2012 02:49:55 +0000 http://www.protocol-online.org/forums/topic/24284-10-mm-na-acetate-buffer-preparation/ http://www.protocol-online.org/forums/topic/24282-getting-rid-of-percipitate-in-ringer-solution/ Fri, 03 Feb 2012 22:17:18 +0000 http://www.protocol-online.org/forums/topic/24282-getting-rid-of-percipitate-in-ringer-solution/ http://www.protocol-online.org/forums/topic/24269-immunofluorescence-for-insulin-receptor/
I permeabilized my cells with Triton for doing immunofluorescence studies for insulin receptor beta (C-19) antibody. If I introduce insulin to these permeabilized cells, I see nice bright fluorescence, confirming presence of insulin receptors. IF I do not add insulin, do you think I will still see fluorescence?]]> Fri, 03 Feb 2012 03:09:58 +0000 http://www.protocol-online.org/forums/topic/24269-immunofluorescence-for-insulin-receptor/ http://www.protocol-online.org/forums/topic/24251-what-does-the-leu7d-selection-marker-do/ Hi, I am a student, I was just wondering if someone could tell me what the Leu7d gene does when used as a selection marker? Wed, 01 Feb 2012 22:23:13 +0000 http://www.protocol-online.org/forums/topic/24251-what-does-the-leu7d-selection-marker-do/ http://www.protocol-online.org/forums/topic/24243-dos-and-donts-for-ph-meter-usage-and-measurement/ What would you all list down as the Do’s and Don’t’s for pH meter usage and measurement ?

As a starter I have listed down few points below…please keep on adding

Do’s

• Ensure that pH meter is properly calibrated before usage
  
• After usage probe should be kept in storage solution

Don’t’s

• dont let the pH probe become dry

please add on more points]]> Wed, 01 Feb 2012 11:43:49 +0000 http://www.protocol-online.org/forums/topic/24243-dos-and-donts-for-ph-meter-usage-and-measurement/ http://www.protocol-online.org/forums/topic/24228-basisprotocols-for-choosing-concentrations-in-testing-medicinal-effect-plant-extracts/ I was conducting a study to test a medicinal effect of a specific plant..I did not see any protocols on what concentration should i used. I decided to use 25%,50%,75% and 100% of the extract but still i should have a basis for using it..Anyone can help me on this...thank you so much.. Tue, 31 Jan 2012 13:57:52 +0000 http://www.protocol-online.org/forums/topic/24228-basisprotocols-for-choosing-concentrations-in-testing-medicinal-effect-plant-extracts/ http://www.protocol-online.org/forums/topic/24218-alternative-for-laf/ to do a sterile microbiology culture,we need help of Laminar Air Flow chamber, There is any other replacement for providing sterile environment for microbial culturing?!!!!!]]> Tue, 31 Jan 2012 01:32:49 +0000 http://www.protocol-online.org/forums/topic/24218-alternative-for-laf/ http://www.protocol-online.org/forums/topic/24177-accuracy-of-ph-measurment-wrt-calibration-points/
what is the measurment accuracy of a sample solution with ph 3.8 by calibrating with buffers pH 4 and 7 or Buffers pH 4, 7 and 10 ??

thanks]]> Fri, 27 Jan 2012 10:26:38 +0000 http://www.protocol-online.org/forums/topic/24177-accuracy-of-ph-measurment-wrt-calibration-points/ http://www.protocol-online.org/forums/topic/24134-dna-extraction-from-plant-tissue-infected-with-fungus/
I have to investigate DNA fragmentation on plant tissue infected with fungus using DNA laddering. This is a new field of investigation for me so I would appreciate any advice. I am especially concern about extraction of DNA from plant sample infected with fungus. Is there any way to distinguish between plant and fungus DNA?
Please help!
Zorana]]> Tue, 24 Jan 2012 13:03:01 +0000 http://www.protocol-online.org/forums/topic/24134-dna-extraction-from-plant-tissue-infected-with-fungus/ http://www.protocol-online.org/forums/topic/24133-southern/ Currently, i'm trying to do some southern to check the number of copy of my transgene. I'm working on wine. After having loads of trouble to get rid of polysacchardides and polyphenol during dna extraction, i'm struggling now with the southern.

I'm working in a small lab and we don't have a lot of facilities and in this case we don't have a stratalinker to crosslink dna on the blot... Thus, i'm trying to do the crosslink with our geldoc. Do you think it's possible or the geldoc is not powerfull enough, because i've got no signal after revealing my southerns...

My other question is, how to force all the digested dna to run in the agarose gel. Because there's a lot of dna remaining in the wells after running...

Thanks for your help]]> Tue, 24 Jan 2012 10:08:34 +0000 http://www.protocol-online.org/forums/topic/24133-southern/ http://www.protocol-online.org/forums/topic/24131-convert/ in reference to oligo probe with MW 17709g/mol

THANX]]> Tue, 24 Jan 2012 08:52:22 +0000 http://www.protocol-online.org/forums/topic/24131-convert/ http://www.protocol-online.org/forums/topic/24118-how-to-convert-5pmolml-to-ngml/ MW is 17709.8g/mol
Length of nucleotides in a probe....50]]> Mon, 23 Jan 2012 09:21:24 +0000 http://www.protocol-online.org/forums/topic/24118-how-to-convert-5pmolml-to-ngml/ http://www.protocol-online.org/forums/topic/24102-conversion-help-for-making-a-solution/
2% A (liquid)
1% B (solid)
.1% C (solid)
.1% D (liquid)
.01 M PBS, pH 7.2

etc. So if I choose to use 40 mL of PBS, then is it correct to calculate the amounts of the other reagents by:
liquid
40 mL PBS * .02 = .8 mL A
solid (the one I'm rather confused on)
40 mL PBS * 1.85 g/mL (sd of .01 M PBS) = 74 g
74 g PBS * .01 = .74 g B

?
Thanks in advance for any help.]]> Sat, 21 Jan 2012 01:59:58 +0000 http://www.protocol-online.org/forums/topic/24102-conversion-help-for-making-a-solution/ http://www.protocol-online.org/forums/topic/24086-mefs-dying-after-passage/
My problem looks like this:
On day 1, I thaw the cells, then plate them on Medium, after they reach confluency, usually on Day 2 I Passage the cells and then majority of them dies or doesn´t attach.
We´ve already used newly prepared Medium, Tripsine and PBS and still no change.. Also sometimes there is a yellow body-like thing in some cultures, but I´ve been told that this is a part of medium, the structures are bigger than cells and once some cells got yellow as well....

Does anyone have any idea what might be happening?

Thank you a lot for trying!!!]]> Thu, 19 Jan 2012 10:57:28 +0000 http://www.protocol-online.org/forums/topic/24086-mefs-dying-after-passage/