我與你

昨日：
當我這天回憶　共你年月裡認識　無盡甜夢你為我織
心靈留下痕跡　是你常令我獲益　漫漫長路有你　讓我懂得愛惜
共經過　數不清的波折　然後又經過　祝福與道別
然後又經過　想不到的轉折　然後再一次相遇時　情是更加熱
天　仍然是藍色　熱愛　無用再著跡　濃情　原是永沒褪色
想到明日回憶　就算　離別你在即　仍然是愛你　一生都衹會這樣說

PCR techniques

Some of the 32 (known?) PCR techniques:
·       Allele specific PCR,
·       Alu PCR,
·       Asymetric PCR,
·       Colony PCR,
·       Competitive quantitative PCR.
·       Competitive RT-PCR (c-RT-PCR).
·       Differential display RT-PCR.
·       Hot start PCR,
·       Immuno-PCR on chip
·       In Situ PCR.
·       Inverse PCR,
·       ISSR PCR,
·       Late PCR,
·       Ligation anchored PCR (LA-PCR).
·       Long PCR,
·       Long PCR,
·       Marathon RNA PCR.
·       Multiplex PCR,
·       Multiplex PCR.
·       Nested PCR,
·       Non-competitive RT-PCR.
·       PCR cloning,
·       PC sequencing
·       RACE-PCR.
·       RAP-PCR (RNA arbitrarily primed PCR).
·       Real time PCR
·       RNase ligase mediated PCR (RLM_RACE PCR).
·       RT PCR,
·       Standard PCR,
·       Suppression subtraction Hybridization PCR (SSH).
·       Touch down PCR,
·       AFLP PCR,
·       Single cell PCR

454 pyrosequencing

As the process continues, the complementary DNA strand is built up and the nucleotide sequence is determined from the signal peak in the pyrogram (intensity).

actual sequence: GCAGGCCT


http://genoseq.ucla.edu/action/view/Pyrosequencing

Amplification Refractory Mutation System (ARMS)

also called allele-specific PCR (ASP)
to amplify or detect mutant DNA, use 3 primers

ARMS has the ability to isolate low levels of a mutant sequence in a background of wild-type DNA. The system depends on the specificity of a primer for the normal sequence and another primer for the mutation.

In ARMS, the primer pair is designed so that one of the 3' ends coincides with a variant nucleotide in the target sequence. When the primer mismatches the template the frequency of extension is very low and consequently the effective number of sequence copies available for amplification is greatly reduced.

The principle of ARMS. Primers are designed to amplify wild-type and mutant sequence. The difference between these two primers is the 3' base, where one matches wild-type sequence and the other the mutant. The reaction containing the mutant primer will only extend mutant template and wild-type template will not be extended by this primer. (3' base of primer is where extension begins, therefore need specificity)

http://www.horizonpress.com/pcr/pdf/rtpcr/rtpcr09.pdf
http://clincancerres.aacrjournals.org/content/12/13/3875/F1.large.jpg

high resolution melting analysis

high resolution melting analysis (HRM):
-used to detect SNP
-must use a saturating dye (e.g. Evagreen)
-after real-time PCR, normalize a melt curve, suppress the curve that differs the most.

Methylation Sensitive (MS)-HRM:

-Bisulfite treatment of DNA converts unmethylated Cytosine to Uracil.
— Methylated Cytosine are protected and remain intact.

-After PCR amplification all Uracils are converted Thymine.

-perform DNA sequence analysis, if T, that means the DNA is unmethylated.
if C, the DNA is methylated.

RNA extraction using trizol

Principle reagents:
guanidinium isothiocyanate (powerful protein denaturant) -> inactivation of RNases
acidic phenol/chloroform(toxic) -> partitioning of RNA into aqueous supernatant for separation

Note: low pH is crucial since at neutral pH DNA not RNA partitions into the aqueous phase.


Nucleic acid (RNA, DNA) partitions in the aqueous phase, while protein partitions in organic phase.

For RNA purification, the pH is kept at around pH 4, which retains RNA in the aqueous phase preferentially. For DNA purification, the pH is usually near 7, at which point all nucleic acids are found in the aqueous phase.


Acid phenol specifically leaves RNA in the aqueous phase.  As the pH decreases, the concentration of protons increases.  DNA carries a negative charge because of the phosphate groups in its sugar-phosphate backbone, which are neutralized in acid by protonation.  In this case, DNA dissolves in the organic phase (like dissolves like).  RNA, on the other hand, is not neutralized in acid because, even though it also has a negative charge, it has exposed nitrogenous bases (it is single-stranded), which can form hydrogen bonds with water, keeping it in the aqueous phase.

http://physiology.med.cornell.edu/faculty/mason/lab/zumbo/files/PHENOL-CHLOROFORM.pdf

thymol gargle

thymol gargle, for mouth wash, anticeptic

hemocytometer

http://celeromics.com/en/resources/docs/Articles/Cell-counting-Neubauer-chamber.pdf

Neubauer Chamber Cell Counting using a hemocytometer

hepatic encephalopathy

7 Dec, 2012
12A ward round
? hepatic encephalopathy
liver dysfunction, toxin affect brain
one of the symptoms: jerking of limbs
order blood test for ammonia level

Reasons to do OGD:
Upper GIB, epigastric pain, anemia