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Hi Mindologist,
I'll go straight to your first question. The first thing that came to my mind is to do what's called the genome walking. Bear in mind that I have not done this (or anything similar) before so someone here might have better alternatives.
Here are some stuffs that could be useful:
www.clontech.com/xxclt_ibcGetAttachment.jsp?cItemId=17326
http://www.protocol-...osts/37995.html
www.ucl.ac.uk/~ucbhjow/b200/Cloning_genomic_DNA.doc
http://www.biotechni...ques-44287.html
https://www.ncbi.nlm...cles/PMC306810/
#147506 Questions about proper technical approach
Posted
on 05 January 2013 - 02:52 AM
#145023 different orientation or direction of genes in expression Plasmid vector
Posted
on 10 November 2012 - 08:03 PM
#141773 SEM analysis of Bacteria
Posted
on 19 September 2012 - 06:23 AM
Hi,
It is necessary* to fix the bacteria (or any cells for that matter) prior to viewing on TEM or SEM. As I'm not really familiar with SEM but the steps/principles running up to viewing is quite similar.
According to the protocol from the University of Oxford:
So we see that it's got nothing to do with bacterial/cell attachment to a surface; rather, serving the purpose mentioned above. Only after fixation will the other steps (post-fixation/dehydration/further treatment/drying/coating) be done.
---
* Unfixed samples are sometimes used negative staining. With unfixed specimens there is the potential problem of changes occurring due to osmotic shock (or to changes in ionic composition) since most negative stains are made up in distilled water. There are safety implications to be considered when examining unfixed bacterial or viral samples.
It is necessary* to fix the bacteria (or any cells for that matter) prior to viewing on TEM or SEM. As I'm not really familiar with SEM but the steps/principles running up to viewing is quite similar.
According to the protocol from the University of Oxford:
Quote
The main purpose of fixation is:
1) to cross-link cellular structures into a matrix so as to preserve the structure of the cells with the minimum of alteration from the living state (i.e. with no changes in morphology, volume, or spatial relationships, and with minimum loss of cellular constituents) and,
2) to protect and stabilize cellular structures from changes during subsequent treatments and from irradiation by the electron beam.
Fixation is the first and most important step in any EM study, since mistakes made at this point render the whole project useless.
1) to cross-link cellular structures into a matrix so as to preserve the structure of the cells with the minimum of alteration from the living state (i.e. with no changes in morphology, volume, or spatial relationships, and with minimum loss of cellular constituents) and,
2) to protect and stabilize cellular structures from changes during subsequent treatments and from irradiation by the electron beam.
Fixation is the first and most important step in any EM study, since mistakes made at this point render the whole project useless.
So we see that it's got nothing to do with bacterial/cell attachment to a surface; rather, serving the purpose mentioned above. Only after fixation will the other steps (post-fixation/dehydration/further treatment/drying/coating) be done.
---
* Unfixed samples are sometimes used negative staining. With unfixed specimens there is the potential problem of changes occurring due to osmotic shock (or to changes in ionic composition) since most negative stains are made up in distilled water. There are safety implications to be considered when examining unfixed bacterial or viral samples.
#139364 Textbook Canon: Expressing Any Genes in Bacteria
Posted
on 13 August 2012 - 02:09 AM
Interesting.
*looks left and right* I think I'll have another go at it.
If all DNA/genes were destined for proteins, then there wouldn't be any tRNAs and rRNAs left. Or RNA I & II, and miRNAs for regulation [in C. elegans].
That should account for the difference in the number of genes and proteins.
I sure hope this is the 'simpler' answer
*looks left and right* I think I'll have another go at it.
If all DNA/genes were destined for proteins, then there wouldn't be any tRNAs and rRNAs left. Or RNA I & II, and miRNAs for regulation [in C. elegans].
That should account for the difference in the number of genes and proteins.
I sure hope this is the 'simpler' answer
#139282 Bacterial adhesion (attachment) to solid surfaces
Posted
on 11 August 2012 - 01:36 AM
Hi,
I'm not sure what kind of samples yours are but in my opinion, the volume in which to submerge/cover/dip your sample would not matter that much, if at all. A search in the literature came up with varying volumes of cell suspension.
On the other hand, you should know how much bacteria (cell concentration) you start with and the amount standardized for all your experiments.
According to this paper, 4 ml of bacterial suspension (2x10^8 cells/ml; E. coli) was used with 8 g of glass beads (0.5-2.5 mm diameter) in round bottomed vials; others used 1 ml for 1 cm2 surfaces. No mention of volume:surface area ratios.
I'm not sure what kind of samples yours are but in my opinion, the volume in which to submerge/cover/dip your sample would not matter that much, if at all. A search in the literature came up with varying volumes of cell suspension.
On the other hand, you should know how much bacteria (cell concentration) you start with and the amount standardized for all your experiments.
According to this paper, 4 ml of bacterial suspension (2x10^8 cells/ml; E. coli) was used with 8 g of glass beads (0.5-2.5 mm diameter) in round bottomed vials; others used 1 ml for 1 cm2 surfaces. No mention of volume:surface area ratios.
#129104 PCR Reaction
Posted
on 14 February 2012 - 03:40 AM
Hi LanMolecular,
Is this your first amplification or a "recipe" passed down to you?
Reason I asked is that the final concentration of Mg2+ (3mM) is quite high for me. For Taq, I usually add it into the individual tubes before starting the cycles. I add the primers into the master mix though.
I have no complains about the cycles and temperature but depending on the outcome, the annealing time [and temperature] might change.
Unless this is a repeat of what was done before, you might need some tweaking after this.
Happy working!
Is this your first amplification or a "recipe" passed down to you?
Reason I asked is that the final concentration of Mg2+ (3mM) is quite high for me. For Taq, I usually add it into the individual tubes before starting the cycles. I add the primers into the master mix though.
I have no complains about the cycles and temperature but depending on the outcome, the annealing time [and temperature] might change.
Unless this is a repeat of what was done before, you might need some tweaking after this.
Happy working!
