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Member since: Jul 28, 2005
From: UK, Scotland
Status: Virology Moderator
My points: 1013    what's this
Name: Jason King

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Aug 24, 2009    01:05 PM

Replied to topic in Immunology forum   MTT assay

You might also want to look at the AlamarBlue solution. Plate out cells in a 96 well plate in their normal medium with 10% AB then follow the fluorescent reduction reaction over time using a plate reader. I usually have samples in triplicate. Works really nicely.

Aug 24, 2009    08:57 AM

Replied to topic in RNA forum   storing rnase

RNase is very stable. Just think how much trouble you go to to ensure that tips and tubes used for RNA work are RNase-free. Freeze-thawing should not be a big problem. The RNase A present in the first buffer used in most DNA plasmid prep kits is stored at 4 degrees and keeps for many months.

Aug 21, 2009    05:27 AM

Replied to topic in Virology forum   Parvovirus tissue culture assays

I had a look an a Parvovirus textbook I have and there is mention of the following cell lines. They allow replication of PPV, undergo CPE within 2-8days and there are good H&E staining and Immunofluorescence assays to detect virus production. 1. Swine Testis (ST) 2. Pig Fallopian Tube (PFT ...

Aug 21, 2009    02:20 AM

Replied to topic in Cell Culture and Tissue Culture forum   LacZ fixation

I had a look back at a paper puplished by a guy I used to work with. He was looking at beta-gal expression in mouse livers and did whole organ fixation and staining. Pattern and load of spontaneous liver metastasis dependent on host immune status studied with a lacZ transduced lymphoma. Kr&uuml ...

Aug 20, 2009    04:01 PM

Replied to topic in Cell Culture and Tissue Culture forum   LacZ fixation

Are these transgenic mice? ie. Should all cells have at least one copy of the plasmid? Or have you administered plasmid to a normal mouse? Which tissue(s) do you expect to have beta-galactosidase expression in? Have you tried doing PCR to detect part of the introduced plasmid?

Aug 19, 2009    05:07 AM

Replied to topic in Cloning (Recombinant Gene Expression) forum   Cloning mystery into pQCXIP

That's interesting. I've had a look at the various prokaryotic methylation target sequences: Dam methylase–methylation at the N6 position of the adenine in the sequence GATC (1,2). Dcm methylase–methylation at the C5 position of cytosine in the sequences CCAGG and CCTGG ...

Aug 18, 2009    11:52 AM

Replied to topic in Cloning (Recombinant Gene Expression) forum   Cloning mystery into pQCXIP

I don't see how your XbaI / EcoRI digest is going to tell you the orientation, unless there is an XbaI in your insert too (in which case you wouldn't need to use EcoRI). From your post it looks like you are saying that there are NotI sites in both vector and insert - is there also a second XbaI? ...

Aug 18, 2009    11:34 AM

I would agree with Ivan when he says that most of the day-to-day technical advice and training is going to come from post-docs. It is vital that your PI ensures that you, the PhD student, really understands the direction in which the project should go. It's always a good idea to read the grant pro ...

Aug 17, 2009    03:44 AM

Replied to topic in Virology forum   Parvovirus tissue culture assays

Are the cell lines you are trying to obtain, already infected with CPV or PPV, or are they to be used to infect with your test samples? If they are not virus infected then it seems strange that there should be import restrictions. Have you already checked whether other Australian labs have the l ...

Aug 16, 2009    05:41 PM

Replied to topic in Cloning (Recombinant Gene Expression) forum   Cloning Problem- colonies only have vector

How are you getting on with the cloning? The above is certainly worth checking but I bet you the problem is that the restriction enzymes are not cutting the ends off of your PCR product, and thus has nothing to do with primers being left over from the PCR. Did your new PCR primer pair work? ie. ...

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