BioForum Bioscience Discussion http://www.protocol-online.org/forums Fri, 10 Feb 2012 01:04:54 +0000 15 http://www.protocol-online.org/forums/topic/24363-xenograft-cells-harvested-from-spleen-of-scidnod-mice/
I'm use to working with cell lines and this is the first time I am working with xenograft cells harvested from the spleen of SCID/NOD mice. I am currently practicing an Alamar blue cytotoxic assay. I bring the cells up from liquid nitrogen and plate them the afternoon before I intend to incubate them with cytotoxic drugs for 48hrs and then adding the Alamar blue and measure the florescence at 0hr and 6hrs. This is a well established protocol by the lab I am currently in. My problem is that the cells in all of my wells (including controls) die before the day when I'm suppose to add the Alamar blue. I think that maybe its my freeze thaw technique that maybe causing the cells to die very quickly. The only criticisms I am getting is that I am really gentle when I bring the cells up from liquid nitrogen. Can someone please give me some tips on how I could be bringing the cells up successfully from liquid nitrogen or does anyone have any insight on what might be the problem??

Thanks.]]> Fri, 10 Feb 2012 01:04:54 +0000 http://www.protocol-online.org/forums/topic/24363-xenograft-cells-harvested-from-spleen-of-scidnod-mice/ http://www.protocol-online.org/forums/topic/24362-nickel-column-purficiation/ We are having problems with the Imidazole that we use for elution in our Nickel Column. We believe that the Imidazole is tearing up our protein. Is there anything else I can use to help elute the Protein, instead of using Imidazole? BTW our protein has a 6X-His tag. Thu, 09 Feb 2012 20:36:29 +0000 http://www.protocol-online.org/forums/topic/24362-nickel-column-purficiation/ http://www.protocol-online.org/forums/topic/24361-weight-of-a-single-lambda-dna/ Can anyone please tell me the weight of a single lambda-DNA molecule of 24000 bp? Thu, 09 Feb 2012 20:11:51 +0000 http://www.protocol-online.org/forums/topic/24361-weight-of-a-single-lambda-dna/ 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/24358-rbs-site-removed-in-construct-will-there-be-expression/

Thanks]]> Thu, 09 Feb 2012 18:37:19 +0000 http://www.protocol-online.org/forums/topic/24358-rbs-site-removed-in-construct-will-there-be-expression/ http://www.protocol-online.org/forums/topic/24357-sarkosyl-and-ni-nta/
I work with a 50KDa his tagged protein (cloned in pET 28 and pET Sumo) that is overexpressed in Rosetta cells. I have a solubility and purification problem.The protein is only soluble (partially) in the presence of 0.3% of Sarkosyl (I've tried different concentrations of IPTG, different temperatures, and different pHs in the lysis buffer). However, with sarkosyl the protein does not bind to Ni-NTA. I've tried to dialyze the lysate but it didnt work. I' ve heard about adding triton and chaps to the lysis buffer to make the protein bind to the colunm in the presence of Sarkosyl. The protein bound indeed, but then it doesnt come out, not even with the addition of EDTA! I already tried to express the protein in pMAL vector, but I had the same problem.

I'm expressing the protein with 1mM IPTG for 3 hours at 37C. My lysis buffer: 50mM Tris-HCl pH 7, 500mM NaCl,0.3% Sarkosyl and protease inhibtors, then I sonicate the cells.

Any of you gurs have an idea of what can I do to purify the protein? I will need the protein for crystallization assays.

Thanks in advance!

Teka]]> Thu, 09 Feb 2012 17:33:26 +0000 http://www.protocol-online.org/forums/topic/24357-sarkosyl-and-ni-nta/ http://www.protocol-online.org/forums/topic/24356-serum-mirna-extraction-small-volumes/
I am new (and maybe a little late) to the miRNA game. I am interested in isolating miRNA only from small serum samples (500 uL to 1 mL).

What is the BEST KIT out there for EFFICIENT isolation of miRNA from serum from limited sample volume?

What is the expected yield? (ng/ uL)

Any modifications to the "pre-existing kit protocols" that would aid in better efficiency?

Thank you,
Maulik]]> Thu, 09 Feb 2012 16:33:59 +0000 http://www.protocol-online.org/forums/topic/24356-serum-mirna-extraction-small-volumes/ http://www.protocol-online.org/forums/topic/24355-where-can-i-rent-20-80liquid-nitrogen-storage-space/
I can't be the first person who wants to put research on hold, but wants to store their precious reagents for future research. Thank you for any ideas!

Bob]]> Thu, 09 Feb 2012 16:16:24 +0000 http://www.protocol-online.org/forums/topic/24355-where-can-i-rent-20-80liquid-nitrogen-storage-space/ http://www.protocol-online.org/forums/topic/24354-depc-treatment-edta-acona/ And Na Acetate?

Hope you can help me!!]]> Thu, 09 Feb 2012 14:56:10 +0000 http://www.protocol-online.org/forums/topic/24354-depc-treatment-edta-acona/ http://www.protocol-online.org/forums/topic/24353-frozen-vs-paraffin-confocal-microscopy/
I was wondering if anyone knows which tissue preperation (frozen vs paraffin) is better for confocal microscopy. Also, I will be using antibodies against phospho-proteins and I have heard that these do no do so well when tissue has been embedded in paraffin.

Any insight would be greatly appreciated!
Thanks,
Carmen]]> Thu, 09 Feb 2012 14:21:28 +0000 http://www.protocol-online.org/forums/topic/24353-frozen-vs-paraffin-confocal-microscopy/ http://www.protocol-online.org/forums/topic/24352-overlapping-pcr-protocol/
I want to make an overlapping PCR with 2 fragments, one has 2200bp and the other has 6000bp. The two fragment share 45 pb homology to their end.

I know that, theorically, I can generate a ''new'' fragment of 8200 bp, but I have no idea of the experimental condition that I should use to get succes! If someone can suggest me some experimental conditions, such as amont of DNA to put into my PCR, PCR amplification conditions and polymerase to use, it would be very helpfull.

thank!]]> Thu, 09 Feb 2012 14:08:25 +0000 http://www.protocol-online.org/forums/topic/24352-overlapping-pcr-protocol/ http://www.protocol-online.org/forums/topic/24350-formaldehyde-cross-linking-problem/
I am trying to optimise ChIP protocol and not able to go pass first step of cross linking!

I am collecting 3-4g plant tissue and crush it in liquid nitrogen. Followed by either storing them in -80 or use it to corsslink in a buffer containing 1% formaldehyde.

I have tried 1% formaldehyde to corsslink my samples (2.5, 5 and 10 minutes). Interestingly, I do see DNA when I De-crosslink but in (IP) control samples which are not de-cross link I dont see even a faint DNA band. Controls without formaldehyde gives good band in de-crosslink or non de-crosslink. This has led me to understand that the samples are over corsslinkned.

1) I am wondering if any one has done cross linking with samples which are first powdered in liquid nitrogen?

2) is 1% formaldehyde ( 37% formaldehyde containing methanol is used) concentration too much for powdered sample?

Cheers for any help in advance!]]> Thu, 09 Feb 2012 12:38:15 +0000 http://www.protocol-online.org/forums/topic/24350-formaldehyde-cross-linking-problem/ http://www.protocol-online.org/forums/topic/24349-petri-dishes-vs-tissue-culture-plates-to-grow-bmdm/
I am trying to harvest the bone marrow and induce the growth of BMDM. However, I have came across many protocols that keep emphasizing the use of non-tissue culture plates (bacterial petri dishes) instead of tissue culture plates to grow them, while other seem to be ok using tissue culture plates.

What difference does it make if I use tissue culture plates instead of the non-tissue culture plate? Is this to eliminate the adherence of fibroblasts or DC ? isnt the addition of m-CSF is good enough to ensure only macrophages will grow by the end of the 6th day? any suggestions on what to use??]]> Thu, 09 Feb 2012 04:47:44 +0000 http://www.protocol-online.org/forums/topic/24349-petri-dishes-vs-tissue-culture-plates-to-grow-bmdm/ http://www.protocol-online.org/forums/topic/24348-to-treat-cells-with-inhibitor/
I just want to ask about some calculations about varying concentrations of drug treatments to my cells. For example, i have a 1uM stock of the drug and I need to treat the cells (3x10^3) in 3 cm dish with 100 nM in 2 ml of medium.

Does that mean i just simply dilute my drug to 100 nM from my 1uM stock and add it directly to my cells? or do i need to compute and consider the 2 ml of medium in the dish?

if you can share with me links or books to do math and computations of concentrations, i would be happy to know. thank yoou...]]> Thu, 09 Feb 2012 02:53:44 +0000 http://www.protocol-online.org/forums/topic/24348-to-treat-cells-with-inhibitor/ http://www.protocol-online.org/forums/topic/24346-propidium-iodide-staining/
Could anyone please tell me what concentration of PI I should use to stain dead cells before running FACS? It seems to vary from protocol to protocol... My stock concentration is 1mg/ml.

Thanks!]]> Thu, 09 Feb 2012 01:03:13 +0000 http://www.protocol-online.org/forums/topic/24346-propidium-iodide-staining/ http://www.protocol-online.org/forums/topic/24345-insulin-receptor-mobility/ What is the mobility of the insulin receptor in the cells? Does it connect to the cytoskeleton or is it freely mobile? Wed, 08 Feb 2012 22:38:11 +0000 http://www.protocol-online.org/forums/topic/24345-insulin-receptor-mobility/ http://www.protocol-online.org/forums/topic/24344-pcr-to-amplify-my-insert-not-working/
Im not used to doing PCR reactions so would appreciate your advice if you have any!

I am trying to change the tag on my plasmid from a Myc to a HA. In order to do this, In order to do this, I digested an empty vector containing the HA tag, and right now, I am trying to amplify my insert with the appropriate oligos.

My mentor has his own oligos for the N term and C term region and I also have a pair so I have been running both at the same time.
In order to run the PCR reaction, I am using a product called "Takara PrimeSTAR HS DNA Polymerase". The protocol they give is pretty straight forward in terms of how much to add in the master mix.

5X PrimeSTAR Buffer - 10 ul
dNTP - 4 ul (Final conc: 200 uM)
Primer 1 - 10-15 pmol (Final conc: 0.2-0.3 uM)
Primer 2 - 1015 pmol (Final conc: 0.2-0.3 uM)
Template - Less than 200 ng
Primestar HS Dna Polymerase - 0.5 ul
MilliQ - up to 50 ul

I run on a gel to confirm that it amplified there after but nothing. No bands. I ran my template, as is, and the band appears, which suggests to me that it is in tact.

Although the protocol suggests running the cycles as

98 degrees 10 sec
55 degrees 5 sec or 15 sec (depending on the Tm)
72 degrees 1 min/kb

for 30 cycles

But my mentor suggested running

98 degrees 10 sec
55 degrees 15 sec
72 degrees 5 min

for 20 cycles
and use 100 ng for template

He says that 30 cycles is too long and that it may lead to more background noise appearing?

If anyone can shed some light I would greatly appreciate it!

Thank you]]> Wed, 08 Feb 2012 22:31:19 +0000 http://www.protocol-online.org/forums/topic/24344-pcr-to-amplify-my-insert-not-working/ http://www.protocol-online.org/forums/topic/24343-which-theory-may-explain-paranormal-phenomena/
John Beloff a well known parapsychologist concluded that PSI occurs becuase of dualism ie the mind and brain are separate. Amit Goswami however in his book “The Self-Aware Universe”, lists some studies on quantum physics that may lead to an explanation of psi that agrees with the theory of a nonphysical and conceptual world. He explains that in quantum physics, objects are not seen as definite things. Instead, objects are possibilities, viewed as something called “possibility waves”. Of course his interpretation due to his research in quantum physics has lead him to formulate idealistic monism, that only consciousness exists in the universe and everything is part of it, he argues against dualism and materialism.

Others however have disagreed and put forward physical and materialistic theories to try and explain PSI.

Michael Persinger claims that much of paranormal phenomena can be explained by low frequency (ELF) electromagnetic waves.

Brian Josephson has claimed that the explanation of PSI may be found in quantum physics. Gerald Feinberg's concept of a tachyon, a theoretical particle that travels faster than the speed of light has been advocated by some parapsychologists who claim that it could explain some PSI phenomena.

Charles Tart however believes PSI is completey non-physical and does not operate to material laws.

There are many theories which try and explain PSI. Which one do you advocate and why? If any?]]> Wed, 08 Feb 2012 22:06:13 +0000 http://www.protocol-online.org/forums/topic/24343-which-theory-may-explain-paranormal-phenomena/ http://www.protocol-online.org/forums/topic/24341-plasmid-extraction/
10 ml culture cell + kanamycin overnight at 37C
observe grow or not
transfer into falcon tube, centrifuge 13000 rpm, at 4C, 10 min
remove supernatant
add 100ul alkaline lysis solution 1, mix the pellet
transfer into apendorf tube
put on ice
add 200 ul solution 2,invert 6 times, put on ice 3-5 min
add 200 ul solution 3, invert
centrifuge 5 min, 4C, 13ooo rpm
transfer supernatant into new tube
add 5 ul RNAse, put in water bath , 10 min at 37C
add phenol ,same volume with supernatant
centrifuge 5 min
transfer the upper layer into new tube
add chloroform , centrifuge, transfer the clear solution into fresh tube (wash 3 times with chloroform)
add 2 volume of alcohol 95%, centrifuge, remove supernatant
add 70%alcohol , centrifuge
remove supernatant, dry overnight
add eluent buffer, store at -21C]]> Wed, 08 Feb 2012 15:58:18 +0000 http://www.protocol-online.org/forums/topic/24341-plasmid-extraction/ http://www.protocol-online.org/forums/topic/24340-hrm-analysis-equipment-requirements/ http://www.bioer.com.cn/productcentere/productcentere?id=001001 do you think it will work for HRM analysis? Thanks in advance!!! ]]> Wed, 08 Feb 2012 15:46:59 +0000 http://www.protocol-online.org/forums/topic/24340-hrm-analysis-equipment-requirements/