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The role is to increase the number of ions in solution to a point where the DNA can be precipitated by the addition of an alcohol primarily. In some extractions such as plasmid preps, it is used to neutralize the alkaline component of the lysis (step 2 NaOH and SDS) and precipitate the proteins and genomic DNA from this step, again through ionic strength.
#155381 Role of sodium acetate in DNA extraction/precipitation
Posted
on 22 May 2013 - 01:02 PM
#152897 SNP genotyping of whole gene
Posted
on 25 March 2013 - 06:29 PM
I used to screen for SNPs in some genes. And share some experience, you have 2 choices:
1.PCR to get whole gene and send with various primers pairs which will be used for sequencing each target SNP. (1 pair of primer for 1 or 2 SNPs which are located near to each other)
2.Design different primers and use PCR to get separate sequences which contain one or more SNPs for each sequence (if they are located near to each other).
You should consider that, the sequencing process cannot be performed well if you have a long sequence and especially if that sequence contains "poly T"
Therefore, you should divide the whole gene into short fragments which contains the target SNPs (by PCR directly or by various primer pairs in sequencing)
Hope this can help you.
1.PCR to get whole gene and send with various primers pairs which will be used for sequencing each target SNP. (1 pair of primer for 1 or 2 SNPs which are located near to each other)
2.Design different primers and use PCR to get separate sequences which contain one or more SNPs for each sequence (if they are located near to each other).
You should consider that, the sequencing process cannot be performed well if you have a long sequence and especially if that sequence contains "poly T"
Therefore, you should divide the whole gene into short fragments which contains the target SNPs (by PCR directly or by various primer pairs in sequencing)
Hope this can help you.
#152814 Suggestions for decorating new lab premises
Posted
on 23 March 2013 - 01:14 PM
hey Ameya...decorating are we now? 
if you want something a bit different, perhaps you can get hold of posters (paintings or lithographs) about the history of science- something like this:
if you want something a bit different, perhaps you can get hold of posters (paintings or lithographs) about the history of science- something like this:
#151992 How 18s gene can be amplified when I use oligo dt for cDNA synthesis
Posted
on 10 March 2013 - 06:31 PM
It is a DNA contamination.
18s is not a good reference gene, because:
1) It has multiple copies in genome. 300 to 400 copies, thus they are much more abundant than target mRNA transcripts, which are usually rare. Thus:
2) You have to dilute sample for 18s as 1:6000 while you dilute sample for other gene 1:20 to bing its CT value between 20-30. Thus:
3) 18s is diluted much more than that of other genes, so it has less inhibitors because of more dilution.
4) It has pseudogenes.
https://www.roche-ap...notes/lc_15.pdf
Check it yourself: goto Primer-Blast website and past both forward and reverse primers in that website and click "Get Primers" buttom.
https://www.ncbi.nlm...s/primer-blast/
You will see that it is not possible to design a pair of primers for 18s that do not bind to more than one place.
5) Ribosomal RNA is resistant to degradation much more than mRNA.
Thus use Ribosomal Proteins instead of 18s. (edited)
http://openwetware.o...R_normalisation
and my suggestion based on my experience:
If you can not find a gene (except 18s )with stable CT value in order to use it as a reference gene, it is your fault.
Because 100% I am sure that you have not done RT, RNA normalization and sampling very well.
-----
Babak
This post has been promoted to an article
18s is not a good reference gene, because:
1) It has multiple copies in genome. 300 to 400 copies, thus they are much more abundant than target mRNA transcripts, which are usually rare. Thus:
2) You have to dilute sample for 18s as 1:6000 while you dilute sample for other gene 1:20 to bing its CT value between 20-30. Thus:
3) 18s is diluted much more than that of other genes, so it has less inhibitors because of more dilution.
4) It has pseudogenes.
https://www.roche-ap...notes/lc_15.pdf
Check it yourself: goto Primer-Blast website and past both forward and reverse primers in that website and click "Get Primers" buttom.
https://www.ncbi.nlm...s/primer-blast/
You will see that it is not possible to design a pair of primers for 18s that do not bind to more than one place.
5) Ribosomal RNA is resistant to degradation much more than mRNA.
Thus use Ribosomal Proteins instead of 18s. (edited)
http://openwetware.o...R_normalisation
and my suggestion based on my experience:
If you can not find a gene (except 18s )with stable CT value in order to use it as a reference gene, it is your fault.
Because 100% I am sure that you have not done RT, RNA normalization and sampling very well.
-----
Babak
This post has been promoted to an article
#137990 Long term storage of RNA
Posted
on 20 July 2012 - 08:32 AM
I thought typical longer term storage was as a pellet in alcohol at -80.
#13775 Important literature for real time PCR users
Posted
on 29 January 2009 - 09:46 AM
Hello,
I have gathered some important documents with many useful information regarding real time PCR.
I have gathered some important documents with many useful information regarding real time PCR.
Attached Files
-
Application_note_Understanding_Ct.pdf 2.03MB
1938 downloads
-
Accurate_normalization_of_real_time_quantitative_RT_PCR_data_by_geometric_averaging_of_multiple_internal_control_genes.pdf 141.4K
1154 downloads
-
Analysis_of_Relative_Gene_Expression_Data_Using_Real_Time_Quantitative_PCR_and_the_2_deltadeltaCT_Method_.pdf 713.66K
1257 downloads
#91190 Why God Didn't Get Tenure
Posted
on 02 November 2010 - 11:55 AM
Why God Didn't Get Tenure
1. He had only one major publication.
2. It was in Hebrew.
3. It had no references.
4. It wasn't even published in a refereed journal.
5. Some even doubt he wrote it himself.
6. It may be true that he created the world, but what has he done since then?
7. His cooperative efforts have been quite limited.
8. The scientific community has had a hard time replicating his results.
9. He never applied to the Ethics Board for permission to use human subjects.
10. When one experiment went awry he tried to cover it up by drowning the subjects.
11. When subjects didn't behave as predicted, he deleted them from the sample.
12. He rarely came to class, just told students to read the Book.
13. Some say he had his son teach the class.
14. He expelled his first two students for learning.
15. Although there were only ten requirements, most students failed his tests.
16. His office hours were infrequent and usually held on a mountaintop.
1. He had only one major publication.
2. It was in Hebrew.
3. It had no references.
4. It wasn't even published in a refereed journal.
5. Some even doubt he wrote it himself.
6. It may be true that he created the world, but what has he done since then?
7. His cooperative efforts have been quite limited.
8. The scientific community has had a hard time replicating his results.
9. He never applied to the Ethics Board for permission to use human subjects.
10. When one experiment went awry he tried to cover it up by drowning the subjects.
11. When subjects didn't behave as predicted, he deleted them from the sample.
12. He rarely came to class, just told students to read the Book.
13. Some say he had his son teach the class.
14. He expelled his first two students for learning.
15. Although there were only ten requirements, most students failed his tests.
16. His office hours were infrequent and usually held on a mountaintop.
#84861 +/- rating system
Posted
on 26 August 2010 - 10:52 AM
pito, on 26 August 2010 - 08:32 AM, said:
Another problem would be the fact that if its like you say then no one will know who pushed the + button.. any fool can push it... or any idiot can push the - button.
...hi gebZ, wasn't Curtis suggesting something similar a while back i.e., to rate the answers/posters (?) like they do in yahoo? This positive feedback and affirming of very good/helpful answers is not a bad idea imho (or in some forums they’d state how many times the poster was thanked)...but if we start implementing this +/- rating then it would feel like an exam every time you give a reply.
And some replies are not outright wrong but just need to be teased out and the more knowledgeable and experienced members can always chip in. This rating system is double-edged: on one hand, it will induce members to come up with well-thought out posts/replies but otoh, this might inhibit some others for even trying esp if they are just throwing ideas around or trying to help out with the little that they know...
and I like pito’s suggestion in case bioforum decides to go ahead with this- there shld be no drive-by rating....whoever rates a post shld identify himself/herself and esp for the *–* rating, they must also state/argue why they deemed the post “wrong”. It becomes more complicated in the end but then it’s more fair and no idiot will start rating posts just for the fun of it....
