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There have been 32 items by bob1 (Search limited from 01-October 19)



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#194685 Complimentary Strand in the 5'-3'

Posted by bob1 on 13 September 2020 - 12:54 PM in -Genetics and Epigenetics-

Note: compliment = saying something nice ("You look lovely today"), complement = paired with A/T, G/C in base terms. Be aware of which you mean - your teacher certainly should know the difference.
 
What the teacher wants depends on the wording of the question - you need to go and read this carefully.
 
There are complementary strands and reverse-complementary strands.
 
The complementary is one where you have the sequence kind of reflected - the exact complement of the top strand. In the example below, the top strand is TTCCAAG, the complement is the one below that, and you would read it in the 3' to 5' direction.
5' TTCCAAG 3'
3' AAGGTTC 5'
 
The reverse complement is similar, but the way you read them is, as you should, from 5' to 3'. In the example below you can see the difference - note the top two are the same as in the example above, the 3rd one is the reverse complement of the first strand, and is the equivalent of mRNA if the first strand is DNA: 
5' TTCCAAG 3'
3' AAGGTTC 5'

5' CTTGGAA 3'



#194668 Datepalm organogenesis

Posted by bob1 on 10 September 2020 - 12:49 PM in General Lab Techniques

Welcome to the site Pete - unfortunately you have come across this forum in its final stages - it was once a thriving forum with many active posters. Those remaining are a tiny minority of the original crew, so you likely won't get much of an answer to your question, unless one of the few people happens to be a plant scientist.

 

Having said that, there are a couple of resources you might not know about, that are good for most protocols:

 

Current Protocols is a great resource of very comprehensive protocols which contain all the materials (and where to get them often) and methods you need for a particular procedure, as well as further reading etc.

Nature Protocols is also a very trusted resource for protocols - they have a plant biology section, which you can search by scrolling down on the linked page until you come to a "Search Nature Protocols" box, then use the pull-down menu to select plant sciences.




#194660 Weird images from algae culture

Posted by bob1 on 08 September 2020 - 05:36 PM in Microbiology

It looks like what you are seeing there are algal cells trapped by surface tension on the surface of the bubble/foam. The clearish object you can see in some of the pictures (e.g. at 10 o'clock position in image 3) looks like an interaction between the light-path and another bubble.




#194645 Weird images from algae culture

Posted by bob1 on 02 September 2020 - 04:50 PM in Microbiology

Which species of alga and scale?




#194611 Light genotyping PCR bands

Posted by bob1 on 21 August 2020 - 03:25 PM in PCR, RT-PCR and Real-Time PCR

It is likely that the contamination is coming from the lab somewhere, basically you re-amplifying already produced PCR products. PCR contamination is a huge issue and very hard to get rid of as it can float around in the air as aerosols (from people opening tubes), it can be on and inside pipettes, on your bench, on the fridge/freezer handles etc. I would thoroughly decontaminate your pipettes as a starting point - bleach works well for this, and use filter-tips, especially for adding template to your reactions.

 

I would doubt that contamination is coming from the clippers, but it certainly pays to at least give them a wipe with ethanol or something between mice. Generally the amount of DNA from an extraction would swamp the stuff coming from small amounts of contamination on the clippers.

 

There are a few ways of getting around this. The most common way is to have dedicated pipettes for use only with PCR products (e.g. loading gels only). Other pipettes are used to set up the reactions, and, if you want to be extra careful, a third set for adding the extracted DNA template. Another additional step is to set up the reactions (without DNA template) in another lab and with pipettes used only for that purpose, where they don't use the same PCRs as you, then take your tubes back to your lab, add the DNA, seal and run the PCR reaction. Make sure that you have a dedicated gel loading area away from your bench.

 

You can also get laminar-flow hoods with UV lights for setting up reactions. It pays to have one for reactions only (no template at all) and one for adding template only, each with dedicated pipettes.




#194603 Light genotyping PCR bands

Posted by bob1 on 20 August 2020 - 01:14 PM in PCR, RT-PCR and Real-Time PCR

Short answer - Generally a known negative should be negative - produce no band at all under any conditions. The fact that you are seeing a band in your known negative under your conditions here indicates that either you have some form of contamination of your sample or a poorly designed primer pair.

 

A quick test would be to re-run the PCR using a completely new aliquot of your known negative and completely new PCR reagents. Set up a few independent reactions (~10 - make them up individually, not from a master-mix of reagents) and see if any of them come up weakly positive. If they do then you have a problem with the PCR itself or perhaps the "negative" isn't actually negative. In this instance it may help to sequence the band from your known negative to see if it is giving a product that matches the positives or a non-specific band.




#194586 HELP: Validation of a polyclonal knock out (KO) cell line when you dont have a g

Posted by bob1 on 17 August 2020 - 01:46 PM in Molecular Cloning

1) My very limited understanding or CRISPR/Cas9 is that it causes targeted cuts in the genome, and because you were making a KO, I assumed that this would be a deletion, because that's what they normally are. If you were using the CRISPR-cpf method then you might be doing insertions. I don't know how you determine the size of the KO, but it is presumably related to the size of your guide RNA. To be conservative I would design primers that give you a product of approx 100-150 bp with the KO, presumably longer without. You should be able to resolve this to single base-pair resolution on a 8 or 12% TBE-acrylamide gel.

 

2) possibly. I don't have experience with CRISPR, so I don't know the details of exactly how it works.




#194572 HELP: Validation of a polyclonal knock out (KO) cell line when you dont have a g

Posted by bob1 on 15 August 2020 - 02:32 AM in Molecular Cloning

1) it depends on how you did the KO.  The % acrylamide (or agarose) depends on how big your PCR product would be with and without the KO.

 

2) Yes you can sequence. If you have a mix of KO and no KO in the polyclonal cells, then you will get a mess from the sequencing if you sequence directly from the PCR. If you run the products on a gel and separate the two potential products, then gel extract the bands and sequence, you should be fine.

 

3) Whole genome sequencing (e.g. MiSeq) might work. The above methods work well and are widely established, they are the methods that were used before CRISPR/CAS was around. You can also use the same methods on RNA rather than DNA.




#194558 HELP: Validation of a polyclonal knock out (KO) cell line when you dont have a g

Posted by bob1 on 13 August 2020 - 05:24 PM in Molecular Cloning

If you know the region of the knock-out, and it is a substantial KO (anything more than say 20-50 bp), you can simply design an array of PCRs - use one primer anchored in the KO region, and one each on either side. The perform 2 PCRs - first with a primer outside, and the anchored inside primer, then second with primers on either side. You should get 5 potential results - no amplification with the anchored primer PCR (if there is amplification, you haven't knocked out, at least not a complete KO). The other reaction will have either a full-length product (for no KO) or a truncated/shorter product (if there is a KO), or a combination of both if it is a chimeric population. It pays to run both PCRs because if the first doesn't work, the second will tell you if you can amplify from that region.

 

This only works if you can separate the different bands on a gel - small KOs will need a greater resolving power gel than you will get with agarose, so look at acrylamide gels for these ones.

 

You can also look for expression of the RNA and see if it is full-length or not, or not expressed at all.




#194508 expressing bacteriophage in mammalian cells

Posted by bob1 on 06 August 2020 - 01:31 PM in Molecular Biology

In addition to mdfenko's answer - it is common to express mammalian viruses from plasmids, often multiple plasmids to make up the genome.  Bacteriophage can, with some modifications for host-cell receptors, insert into a mammalian cell and express genes from mammalian derived cassettes (e.g. here (PDF)).

 

However, in the initial question you asked:

 

 

Is it possible to express bacteriophage in mammalian cells by codon optimization? Any relevant literature? thanks in advance.

The short answer is: no, because the bacteriophage has a bacterial expression system that is not utilized by mammalian cells. If (as in the linked paper) you can insert a mammalian promoter, then you should be able to express some or all of the proteins needed, but this does not necessarily equate to assembly as you need the right components in the right locations at the right time for bacteriophage assembly. In bacteria which only really have one cellular compartment this isn't a problem because all the components are in proximity to each-other, but mammalian cells have many compartments (e.g. golgi, nucleus, endoplasmic reticuluum, etc.). Each of these compartments have assigned roles such as protein generation (ER), DNA replication (nucleus), which would separate the functions of each out so that while capsid might be produced, there wouldn't be any DNA to go into it or vice-versa.

 

I think you are also misunderstanding what codon-optimization is. It is a system to make the expression of genes more amenable to protein production in mammalian cells by altering the DNA so that forms of the codons used are the ones more commonly used in mammalian cells. Just because you codon optimize something doesn't mean it will express in a mammalian cell, you also need mammalian promoters and terminators, as well as a means of getting the DNA into the cell in the right orientation respective to the promoter. There are other components that can help too such as the Kozac sequence.




#194466 end point PCR using real time PCR machine (QS5)

Posted by bob1 on 26 June 2020 - 02:33 PM in Molecular Biology

Yes, it should be fine. It's just DNA, so it is quite stable at -20.




#194463 Senescence.... positive result?

Posted by bob1 on 24 June 2020 - 07:09 PM in Cell Biology

I know this is late - yes, it looks like a lot of the cells that have stained blue are senescent. I think normally when you do this assay you would use it quantitatively in a spectrophotometer/plate reader.




#194461 end point PCR using real time PCR machine (QS5)

Posted by bob1 on 24 June 2020 - 07:04 PM in Molecular Biology

Yes, any PCR machine can do end-point PCR... in fact every PCR you do is an end-point PCR because the end-product (DNA band specific for your sequence) is the same whether you are doing qPCR or conventional PCR. In fact, you can even take a qPCR product and run it on a gel to check for single bands. This is how it was done before the days of melt-curves.

 

There is no need to deselect dyes or anything else, these don't interfere with the production of the DNA.




#194365 How to statistically/bioinformatically measure the contribution of mutations to

Posted by bob1 on 25 May 2020 - 07:07 PM in Bioinformatics and Biostatistics

Principal component analysis? Depends on what sort of stats you have on each one - this is way beyond my understanding of stats, so you might want to talk to a proper statistician if you can.




#194329 ELISA a biosensor?

Posted by bob1 on 18 May 2020 - 01:35 PM in ELISA and Immunoassay

I think you need to look up what the definition of a biosensor is and how an ELISA works, then tell us whether you think ELISA is a biosensor or merely a biochemical reaction detected by a machine.




#194328 Can't remove Ponceau staining!

Posted by bob1 on 18 May 2020 - 01:33 PM in SDS-PAGE and Western Blotting

You can also wash the Ponceau off with a blocking solution - milk powder in buffer or BSA in buffer work well.




#194297 formal name for a type of repeat

Posted by bob1 on 11 May 2020 - 04:13 PM in Bioinformatics and Biostatistics

The term invariant fits the description of what you want. Here's an example in a scientific paper.

 

The only time you might find such a situation in real life is where you have a string of amino-acids that can only start with one nucleotide. If you refer to a genetic code table, you will see that both Phenylalanine (F) and Tyrosine (Y) are two amino acids that only ever start with T/U in the first position. So assuming no mutations, every first position in a string such as:

Y.  Y.  Y.  Y.  Y.  Y.  Y.  F.  F.  F.  F.  F
Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Phe Phe Phe Phe

Could have the DNA sequence:

TAT TAC TAT TAC TAT TAC TAT TAC TTC TTT TTC TTT TTC TTT
Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Phe Phe Phe Phe Phe Phe

Where every 1st position in a codon is a T - these would be invariant nucleotides in this case. In real life you are unlikely to empirically find such a case, and if you do, it is most likely to be a single base that can only ever have 1 amino-acid configuration for some reason.

 

 

 




#194206 Order of lab manipulations (displaced topic)

Posted by bob1 on 23 April 2020 - 03:13 PM in -Microbiology and Virology-

Can you transcribe the actual question - it looks like you are trying to distinguish between cocci and bacilli with biochemical/staining tests?

 

If this is the case, think about how you would grow the organisms (which agar and what does that agar select for/against), then think about what result each organism will give with what test. These answers will be in the course notes somewhere - and certainly on the internet with some googling.




#194150 Question about tetracyclin

Posted by bob1 on 20 April 2020 - 02:19 PM in General Lab Techniques

Degradation and melting are two different things - in solution tetracycline will degrade quite quickly in the absence of heating. As far as I know, you can not heat tetracycline in solution and expect it to work after heating.

 

Now, I have no idea if you could heat the crystalline form to melting point and if it will stay stable after that, but I suspect it would not work as well as it would before melting.




#194117 Question about tetracyclin

Posted by bob1 on 19 April 2020 - 02:17 PM in General Lab Techniques

No - melting temperature does not equate to stability. Tetracycline is particularly unstable in aqueous solution, it will last a few days.

 

You can find information about stability from most suppliers - it should be on the data-sheet.

 

Here's one such sheet I found and the relevant information, note the 3rd sentence (bolded by me):

 

 

Tetracycline hydrochloride is stable if stored in a dry place and protected from light. It is water-soluble with approx. 11 mg/ml at 28°C. The stability in solutions is optimal at pH 3 - 5.2 (shelf life approx. 6 - 12 days). It may be stored between -20°C and 37°C. Since Tetracyclin is light-sensitive, solutions and agar plates containing TC should be protected from light. The recommended working concentration is 10 - 50 μg/ml, the stock solution e.g. 1.25 mg/ml. Magnesium ions antagonize with the activity of TC.




#194101 Terminology: chylotrophic

Posted by bob1 on 19 April 2020 - 02:15 AM in Microbiology

I don't think there is any comparable word, though a description might be "external digestion". From the form of the word I would have guessed that it meant something like moving in the direction of (a la tropism) chyle, not the movement of chyle.




#194027 Terminology: chylotrophic

Posted by bob1 on 17 April 2020 - 05:21 PM in Microbiology

Not commonly used, I consider myself fairly literate and didn't know the term




#185655 polar and non-polar substances in one mix

Posted by bob1 on 08 April 2020 - 02:22 AM in General Lab Techniques

Dimethyl sulfoxide is the commonly used one.




#184422 Viral isolation kit QIA buffer substitute

Posted by bob1 on 07 April 2020 - 12:43 PM in PCR, RT-PCR and Real-Time PCR

Yep, same problem world-wide. Have a look at the MSDS; it tells you that this is indeed a guanidinium salt solution. I think from memory there is a buffer (tris probably), some EDTA and a couple of other things, but I don't know the exact composition.

 

As far as I can tell, the columns are the same for pretty much every kit and the buffers are just based off the old manual RNA extractions that we performed 20 years ago. If you have access to a recent copy of Sambrook's Molecular Cloning, it'll probably tell you explicitly how to make these. Unfortunately I only have a 1989 version and only Vol. 1 and 2, so I can't even look it up for you.




#182468 ?what is better for protein precipitation ammonium acetate or sodium chloride in

Posted by bob1 on 21 March 2020 - 01:17 PM in Molecular Biology

@iosman987 - I think you answered the wrong question...





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