Lipid extracts should not be stored in the dry state.

Oxidative degradation is slower in solutions even in the absence of added antioxidants; This protection depends on the chemical nature of the solvent and the physical conditions of storage. Air and light should be avoided. The best storage conditions are a super freezer (- !"#$ to store for a long time (up to one year$ lipid extracts in chloroform-methanol mixtures filling well stoppered glass vials (with Teflon liners$% the cap being secured with a wide length of self-stic&ing tape. 'encil written labels should be protected by an outer layer of polyester tape. (or long period of storage% it can be useful to flush vials or tubes with nitrogen before closing to prevent fatty acid oxidation. (or the same purpose% a low amount of antioxidant such as )*T (butylated hydroxytoluene or +%,-di-tert-butyl---methoxyphenol$ or ethyl gallate (i.e. about .! /g )*T0ml$ may be added in the solvent if the natural antioxidant amount is estimated too low (purified extracts$. The antioxidant is used as a concentrated solution in ethanol (1! mg0ml$. After long periods of storage% the cleavage of ester lipids and plasmalogens must be expected with a concomitant rise in free fatty acids% diacylglycerols methyl or ethyl esters. These problems can be minimized by using neutral extracts and +-propanol instead of methanol or ethanol in the solvent mixture. 2t must be remembered that best results are obtained with freshly prepared materials. To prevent labile lipids from oxygen attac& it is possible to use a commercially available oxygen absorber (Ageless% 3itsubishi 4as #hem #o$ placed inside the sample pac&age. After a short 5pretreatment5 at +"#% the labile lipids appear preserved even at room temperature for a long time. This method allows transportation of biomaterial samples to a distant laboratory without utilizing any freezing system (Hirao S et al., J Oleo Sci 2003, 52, 583$. 2t has been demonstrated that after one year storage of plasma samples at -6!"# various lipid parameters (cholesterol% triglycerides$ were altered (Devanapalli B et al., Clin Chim Acta 2002, 322, 17 $. Thus% even at low temperature% intact biological samples must be extracted as soon as possible. 2n contrast% it was shown that after storage at -6!"# up to - years% the fatty acid compositions of plasma triglycerides and erythrocyte phospholipids were practically unchanged (Ho!"on # et al., Clin Chim Acta 2002, 321, $3$.

DNA Storage and Quality

Monday 22 August 2011 Product types: Chromosome-specific arrays, Custom arrays Reliable measurement of !A concentration and purity is important for many applications in molecular biology, especially array comparati"e genomic hybridisation #aC$%& 'here accurate determination of !A concentration is critical( )mpurities in !A can lead to inaccurate measurement of !A concentration and could potentially inhibit subse*uent labelling reactions( +his article pro"ides recommendations for measuring !A *uality for use in aC$% based on our e,perience of running thousands of samples per 'ee- in our highthroughput genomic ser"ices laboratory( )n addition, detailed information is pro"ided on the theory and practice behind measuring !A purity and concentration(

Introduction
+he preser"ation and storage of !A is of interest to scientists in a 'ide range of fields and disciplines( .hen considering 'hat is meant by the terms /preser"ation0 and /storage0, it must be remembered that the perspecti"es of these scientists 'ill "ary considerably( 1or e,ample, !A re*uired for testing a pharmaceutical product 'ill need to be stable for a fe' years, 'hereas samples used in e"olutionary biology ha"e been in e,istence for millions of years1( +he do'nstream applications in 'hich these samples may be used are "ery different and therefore the minimum *uality and *uantity of !A re*uired 'ill "ary( Although nuclease contamination must al'ays be carefully a"oided 'hen handling !A, it is chemical degradation that represents the ma2or threat to !A preser"ation(

DNA storage strategies

)n general, there are four broad strategies for long-term !A preser"ation:

Room temperature on a /dry0 solid matri, 3204C 3504C 31674C #storage in li*uid nitrogen&

+'o of these methods, dried and stored at room temperature and storage at 31674C, share a common mechanism 'here the !A is maintained in a glassy #or "itreous& state( )n the glassy state, molecules lose the ability to diffuse such that the mo"ement of a proton is estimated to be appro,imately one atomic diameter in 200 years, thereby pre"enting chemical and nuclease degradation( )f moisture is added to the /dry state0 or the temperature is raised abo"e the glass transition temperature of 'ater, mo"ement and reacti"ity of protons is reestablished and damage to the !A can occur2(

The glassy state

$lass is a state of matter8 glasses combine some properties of crystals 'ith some properties of li*uids( $lass formation, or "itrification, is the creation of a li*uid solution 'ith the "iscosity of a solid( $lasses can be formed either by increasing the solution concentration or by lo'ering temperature( )n fro9en a*ueous samples, glasses are formed by a combination of the t'o( $lasses are usually supersaturated and thus metastable, but the high "iscosities and acti"ation energies re*uired for phase separation may pre"ent decomposition for long periods, the duration being dependent upon composition and temperature( +he formation of glasses is normal for substances that remain li*uid o"er a 'ide temperature range #the :good glassformers:& and can be induced for most li*uids if cooling is fast enough to bypass crystalli9ation( uring reheating, but still belo' the melting point, good glassformers e,hibit glass transitions as they abruptly transform into supercooled li*uids, 'hereas other substances transform directly from the glassy to the crystalline state;( <torage at 3204C to 3504C may 'ell pro"ide ade*uate conditions depending on the *uality and *uantity of !A desired and the time frame in 'hich the sample 'ill be stored( %o'e"er, neither of these conditions 'ill maintain !A *uality e*ui"alent to maintenance at li*uid nitrogen temperatures o"er e,tended time periods #e(g( decades&( )n contrast to storage of !A in solution at "ery lo' temperatures, it is also possible to store !A dried( +his can be a practical alternati"e for long-term storage( )n addition to reducing molecular mobility, dehydration also remo"es 'ater that can participate in hydrolytic reactions( +here are se"eral methods of remo"ing 'ater from li*uid preparations8 these include spray drying, spray free9e drying, air drying or lyophilisation( <praying !A is perhaps the least popular option as it has been associated 'ith damage introduced by shear stress( Another option for storage of dried !A is on 1+A= Cards #.hatman&( Cells are lysed upon application to the card and the nucleic acids are immobili9ed( <tudies ha"e sho'n that genomic !A that has been stored on 1+A Card at room temperature for o"er 1> years has been successfully amplified by PCR?( +he cards are supplied 'ith a reagent 'hich enables high molecular 'eight !A to be released from the matri, for use in many molecular biology techni*ues( Although !A stored on 1+A Cards may be suitable for microarray studies, this has not been tested by @$+( )n a laboratory setting, !A is most commonly stored at ?4C, 3204C or 3504C( +o a"oid chemical and en9ymatic degradation, !A is often stored as a precipitate in ethanol at 3 504C( Ander these conditions, nucleic acids are stable for prolonged periods, but must be isolated from the ethanol, transferred to a*ueous buffers, and typically *uantified prior to use( +hese manipulations render ethanol precipitations undesirable for applications 'here the samples are needed on a regular basis( A*ueous solutions of !A 'ould be the most con"enient, but nucleic acids are sensiti"e to depurination, depyrimidination, deamination and hydrolytic clea"age, 'hich limit prolonged storage under these conditions( )t is possible to inhibit these acid-catalysed degradation processes by storage in al-aline solutions( +he ionic strength of the solution 'ill also affect depurination rates, so storage in salt solutions as opposed to a lo' ionic strength buffer 'ill help( Assuming the absence of nucleases 'hen !A is stored in a saline solution 'ith p%5(B, the most common form of damage is "ia o,idation( +he rate of o,idation is enhanced by the presence of trace metals #e(g( 1e;C, Cu2C& due to the production of free radicals "ia 1enton-type reactionsB(

+he rate of o,idation is enhanced by the presence of trace metals #e(g( 1eDC, CuEC& due to the production of free radicals "ia 1enton-type reactions?(

The Fenton reaction

+he 1enton reaction 'as disco"ered in 156? by %(F(%( 1enton( +his reaction describes the brea-do'n of hydrogen pero,ide by metal ions such as iron( +he e,act mechanism is still to be elucidated but, broadly spea-ing, the reaction can be described as: 1eEC C %2@2 G 1eDC C (@% C @%1eDC C %2@2 G 1eEC C (@@% C %C +he highly reacti"e free radicals that are produced during this reaction 'ill go on to react 'ith other compounds and can be a ma2or cause of damage to !A or other nucleic acids7( Contamination by transition metals can increase o,idation le"els8 therefore, de-metalation of all components # !A, buffers, and 'ater& can significantly reduce degradation during storage( Anfortunately, is it impossible to measure "ery lo' le"els of metal contamination or to completely remo"e trace amounts( %ighly purified clinical grade !A can contain iron le"els of ;03?0 ppb, 'hich is 'ell abo"e the safe le"el of B ppb( A solution could be to incorporate chelators into !A preparations to limit metal-catalysed reactions( Hut it should be remembered that chelating agents can inhibit nucleases 'hich can ha"e an ad"erse effect on do'nstream reactions( Also chelation of metals does not completely pre"ent 1enton-type chemistry and it may be ad"antageous to include antio,idants or sca"engers in the storage medium( A study by Iiagen> sho'ed that, follo'ing e,traction using their I)Aamp= !A Hlood Mini Jit, !A 'as stable for at least 5 years8 ho'e"er, the *uality of the !A 'as dependent on the temperature and the buffering conditions used( !A eluted and stored in Huffer AK #10 mM +risLCl8 0(B mM K +A, p% 6(0& 'as stable for at least 5 years at either 3 204C or 2354C( !A samples stored in 'ater for 5 years remained intact 'hen stored at 3 204C, but 'ere degraded to "arying degrees 'hen stored at 2354C( +he degradation of the !A stored in 'ater at 2354C may ha"e been due to acid hydrolysis #at p% B37&, since 'ater is unbuffered and can be slightly acidic( Alternati"ely, the !A may ha"e been eluted 'ith 'ater from a 'ater source contaminated 'ith minor traces of nucleases, or the !A may ha"e been contaminated 'ith nucleases during the annual opening of the sample tubes( Huffer AK, supplied by Iiagen is intended to protect !A during storage, 'ith the +ris buffering against lo' p% and the K +A inhibiting nucleases( )f e,tracting !A from 'hole blood using the $entra Puregene= Hlood Jit, reports from Iiagen sho' that it is preferential to collect the sample in K +A tubes to reduce !ase acti"ity5( %eparin can be used but is not ideal as it is thought to bind to !A during purification( <tudies ha"e sho'n that blood can be stored for up to 2? hours at room temperature prior to purification but, if possible, it should be placed on ice( 1or longer term storage, blood should be stored at ?4C or 3504C, studies ha"e sho'n that good *uality !A can be obtained from blood stored at 3504C6( <torage at 3204C is not recommended as this can result in lo'er yields( +he tha'ing temperature can also ha"e an effect on !A *uality

and Iiagen recommend that samples are tha'ed *uic-ly at ;>4C rather than slo'ly at room temperature10( +here is some contro"ersy regarding !A damage resulting from repeated cycles of free9etha'ing and it is common practice to store !A in ali*uots to minimise the number of times the !A is tha'ed( )nterestingly, a study by Hethesda Research Maboratories11 found no e"idence that free9e-tha'ing cycles causes !A damage( +hey made the obser"ation that pre"ious studies e,amining the effect of repeated free9ing and tha'ing used radio-labelled !A and the radioacti"e label itself 'as causing damage to the !A rather than the number of free9e tha' cycles( %o'e"er, as a cautionary note, this study utilised high *uality !A( <toring ali*uots of !A 'ould still be beneficial in order to minimise degradation, 'hich could occur at higher temperatures due to impurities, and to a"oid contaminating the main !A stoc-(

Summary
+o ensure high *uality microarray results, 'e recommend the follo'ing !A storage strategies:

<hort-term storage #'ee-s& at ?4C in +ris-K +A Medium-term storage #months& at 3504C in +ris-K +A Mong-term storage #years& at as 3504C as a precipitate under ethanol Mong-terms storage #decades& at 317?4C or dried

Oil Palm
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