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  • Baculovirus Generation
    Baculovirus Generation
    Baculovirus GenerationViable cell count1) dilute 5 uL Viacount Flex w/ 995 uL culture media2) mix 10 uL cells w/ 190 uL diluted Viacount3) measure cell count and viability in GuavaTransfection1) In a sterile 96 well plate, add:a. FASTBAC = 5 uL DNA / wellb. pBAC = 1 ug shuttle vector + 1 uL linearized DNA2) Make master mix of Fugene HD in Transfection mediaFor each well:3 uL Fugene HD100 uL Transfection media3) Add 103 uL of Fugene HD mix to each well4) Incubate at room temp in laminar flow hood for 30 min5) Aliquot 2.5 mL Sf9 cells (cell density = 1x10e6 cells/mL) into 24 well block6) Transfer 100 uL DNA:Fugene mix from 96 well plate to cells in 24 well block7) Seal block and transfer to shaker8) read GP64 assay @ 4 days post-transfection and set up 5 mL expression blockGP64 assay1) mix 10 uL cells + 10 uL anti-GP64-phycoerythrin Ab (1:100 in 1x TBS + 4% BSA)2) incubate >20 min @ 4⁰C in the dark3) dilute samples w/ 180 uL 1x TBS4) Open Cytosoft 5.35) Click Guava Express Plus6) Open Work Edit 5.37) Select wells containing samples8) Check:Events = 2000dilution factor = 209) Save worklist wile in GE WORK LIST10) Return to Cytosoft 5.3 and click "START WORKLIST"11) load tray- match A1 to A1 marking12) Select worklist "GE WORK LIST"13) Click "RETRIEVE SETTINGS"- in LOCAL DATA TRANSFERS folder -> SETTINGS FOR GP64 .. -> GP64_PE14) Click any well containing sample to run15) Click next step when histogram peak increases16) Click resume to read entire plate5 m Expression1) Add 5 mL cells (~2.0x10e6 cells/mL) / well in a 24 well block2) Add 50 uL transfected cells to each well3) seal block with easybreathe seal and incubate @ 27⁰C in shaker4) Read GP64 @ 24 h post-infection5) Read FLAG-FITC @ 48 h post-infection- similar to GP64 assay except:a) mix 10uL cells + 12uL anti-FLAG-FITC Ab + 7AAD +/- Triton X-100b) run settings for FITC-7AAD
    자연과학| 2014.09.13| 2페이지| 1,000원| 조회(63)
    태그 generation, Baculovirus, Baculovirus Gener..
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  • Manual of X-ray crystal diffraction processing using HKL2000
    Manual of X-ray crystal diffraction processing using HKL2000
    1. 프로그램의 시작- login-프로그램 실행: shell에서 HKL2000을 치거나 바탕화면에서 아이콘을 클릭한다.-실험한 Beamline을 선택-Data IndexIntegrateScale순으로 processing을 한다.2. Processing (Native data)1) “Data” 대메뉴를 선택한다.(1) SAD, native중 native를 선택한다.(2) data를 불러오고 processing 후 data를 저장하는 옵션을 설정한다.- [Directory tree]메뉴 에서 data가 있는 directory를 찾는다.- [Load/Create New Set]메뉴에서#1: directory를 지정하고 New raw data dir에서 >>을 click 하면 data path가바뀐다. – processing 할 data 위치 지정#2: data가 있는 directory에서 를 눌러 output이 저장될 directory를 만들고 지정한다. (Data가 저장되어 있는 directory 밑에 “x”라는 이름으로 만든다.) Output data dir에서 >>을 click 한다. –processing 한 data의 저장위치 지정#3: 를 선택하면 새로운 창이 뜨면서 data set이 표시된다.(3) “Select data sets from the list”에서 processing 할 data image를 선택- 선택이 잘되면 data의 정보가 표시되는 창이 안에 생긴다. 여러 개가 있다면 를 눌러 선택# Data를 얻고 있는 도중이라면 data가 나타나는 하늘색 창에 start 와 end에 실험이 끝난 image가 표시가 된다.# Data Processing을 평가하기 위하여 1-50장만을 processing 하기 위하여 [Set Controls] 메뉴의 를 선택하고 “Number of Frames”를 50으로 바꾸면 된다. 그러면 processing data 정보가 start: 1, End: 50으로 바뀐다.# 실험도중 data를 processing한다면 끝나는 마지막 image의 수를 “Number of Frames” 항목에 넣고 processing을 시작을 하면 data수집되는 데로 자동으로 processing이 진행된다.2) “Index” 대메뉴를 선택한다.: crystal의 symmetry를 찾고 unit-cell을 찾는 과정임. 여러 개의 data가 있다면 “pending sets”를 확인하고 올바로 지정할 것(1) [Controls] 메뉴에서 를 선택한다. -새로운 창이 뜨면서 X-ray diffraction 결과가 나타난다.(2) [Display]창에서 를 선택한다.-선택-peak확인- 또는 으로 조절-peak 확인-상황에 따라서 이용하여 peak 선택* peak search를 하면 diffraction결과가 있는 창에서 붉은색 원이 peak 위에 그려진다. 처음에는 기본적인 옵션으로 peak search가 이루어진다. 좀더 많은 peak을 search 하거나 줄이고 싶으면 이나 을 선택하여 조절한다. 조절하여 원하는 peak을 선택하였으면 를 열고 spot과 peak search의 영역이 올바르게 선정되었는지 확인 한다. Spot size의 조절은 mosaicity와 χ2에 영향을 준다. 자동으로 peak을 search한 후 more peak이나 fewer peak으로 조절을 했음에도 불구하고 low resolution 지역에서 peak이 지정되지 않았다면 를 선택하여 peak을 추가시켜서 진행해야 한다. Reference peak이 모자라면 space group을 정하는데 문제가 생길 수 있다.(3) [Display]창에서 를 누른다.(4) [Refinement Option]에서 메뉴에서 을 선택한다. -이 옵션을 안 하면 error 보정이 안 된다.(5) [Controls]에서 “ on set 1” 버튼을 누른다. -실험을 한 모든 이미지가 processing(indexing이라는 표시가 깜박임)이 진행되고 결과가 출력된다.# Bravais Lattice Table 결과 설명: 새로운 창 (Bravais Lattice Table )이 열리면서 결과가 출력된다. 아래에서부터 위로 높은 symmetry가 위치한다. Symmetry이름, difference, data로부터 계산 값이 출력되고 위에는 계산된 값 밑에 바로 symmetry에 따른 이상적인 값이 출력된다. Difference(%)가 적은 순에 따라 녹색, 남색, 붉은색으로 출력이 된다. Symmetry의 선별법은 difference가 적으면서 높은 symmetry를 가지는 것을 선택한다.이러한 방법으로 indexing이 끝난 후 [Controls]메뉴의 를 열어 선택한 symetry의 difference가 0%으로 떨어졌는지 확인 한다. 적절한 선택이었다면 0%로 된다.△priothl….. 20% 113.3 122.3 122.4 ………………. (계산값)113.2 120.6 123.4 ……………….(lattice의 계산값)(6) [Bravais Lattice Table]창에서 위의 조건에 만족하는 Symmetry를 선택한다.(7) [Bravais Lattice Table]창에서 선택(8) [Controls]메뉴에서 을 누른다.(9) “Refining”이라는 붉은 글씨가 깜박이며 refine을 실시되고 [Display]창에는 refine에 의해 찾은 peak이 나타난다. 끝나면 “Ready”로 표시된다.(10) Refine을 실시하면 peak search로 찾은 것과 같은 노랗거나 Cyan이거나 붉은 원이생긴다. 이때 [Display]창에서 를 열고 창에서 를 누르면 box size (네모)와 spot size (원)이 나타난다. 이 원과 박스를 [Integration Box]에서 적절하게 조정한 후 refine을 다시 실시한다.. 원은 spot을 정하는 것이고 박스는 background의 값을 정하는 것이다. 박스는 겹쳐져도 괜찮으나 spot을 지정하는 원은 겹치면 안되므로 영역을 적절하게 조정한다. 조정한 후 다시 을 누른다. 결과 값에서 χ2가 안정화가 되어 더 이상 변하지 않으면 끝난 것임. χ2값은 낮을수록 좋음.(11) [controls]메뉴에서 을 누른다.# 을 누르면 “integrate” 메뉴로 이동한다.3) “Integrate” 대메뉴에서 Integration을 실시한다.(1) [Controls]메뉴에서 을 누른다.# 를 누르면 “Integrating”이라는 글씨가 깜박인다.4) “Scale” 대메뉴에서 Scaling을 실시한다.Space group을 선택한다. 보통 이미 들어가 있다.에서 사용할 Resolution을 선택한다. : min:50A, Max: 2.0A: Small slippage imperfect Goniostat를 선택한다.# resolution을 제외한 옵션은 모두 default(원래 들어가 있는 값)을 사용한다.[Controls]메뉴에서 를 눌러 scaling을 실시한다.# [Controls]메뉴위에 “Scaling”이라고 깜박이다가 끝나면 “Done”이라는 메시지가 표시된다.끝나면 을 열어 log file을 확인하고 data를 평가한다.5) Log file을 통한 data 결정 및 processing 방법 결정(1) Log file을 열고 file의 맨 아래 있는 “Summary of reflections intensities and R-factors by shells”의 항목을 보고 판단한다.(2) 아래의 기준에 맞는 resolution을 잡아 scaling을 다시 실시한다.# 기준 : I/error = 3, Linear R-factor = 40%이하# I of sigma: Intensity 와 noise의 대비를 말한다. 이 값을 통하여 어떤 intensity의 spot 을 선별 할 것인가를 정한다. Intensity는 noise의 2배가 되는 점을 선택한다.# R factor: R-factor가 40-50%가 되는 것을 선택한다.# 위의 두 가지를 모두 고려하여 scaling할 resolution을 선택한다. -본 실습의 경우 선택한 resolution은 min 50, max 1.8을 선택함. 조교의 말에 의하면 completeness를 손해 보면서 resolution을 높인 것과 resolution을 포기하면서 completeness를 높인 것 등 여러 가지로 processing을 하여 분석에 이용하는 것이 좋다고 하였음5) 위의 기준으로 다시 scaling 실시
    자연과학| 2014.09.13| 4페이지| 1,000원| 조회(103)
    태그 processing, Manual of X-ray crys, Native d..
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  • Primer extension
    Primer extension
    Primer ExtensionIntroductionprimer extension analysis is utilized to quantitate mRNA levels, and to detect low abundance mRNA species. In addition, primer extension analysis can also be utilized to map the 5'-end of transcripts to determine the exact start site(s) for transcription.- Primerthe ideal primer is a short single-stranded DNA fragment that is complementary to a region within the 5'-end of the transcript to be analyzede and is approximately 20-40 nucleotides in length.the best results when targeted to region within 150 nt of the 5' terminus of mRNA oligonucleotide primers should have a GC content of ~50% and should have a G or C residue at the 3' terminus.- Annealing temperatureIn most cases, where the primer is ~50% GC content & 20-30mer length, the optimum annealing temperature between 40℃ and 60℃Materials- Buffers and solutionsAmmonium acetate (10 M)ChloroformDithiothreitol (1 M) : strong reducing agentEthanolFormamide loading buffer80% deionized formamide10 mM EDTA (pH 8.king primer extension mix-> store the actinomycin D stock solution at -20℃ in the darkSodium acetate (3 M pH 5.2)TE (pH 7.6) - 10 mM Tris-Cl (pH 7.6) + 1 mM EDTA (pH 8.0)Trichloroacetic acid (1% and 10% TCA)-> dilute 100% stock solution just before use. chill working solutions in ice- Enzynes and BuffersPolynucleotide KinaseProtein inhibitor of RNaseReverse transcriptase- GelsDenaturing polyacrylamide gel containing 8 M urea- Nucleic Acids and Oligonucleotidescarrier RNA (yeast tRNA)DNA markers, radiolabled, for gel lectrophoresispoly(A)+ RNAoligonucleotide primer- Radioactive Compounds[?-32P]ATP (10 mCi/ml, 7000 Ci/mmole)- Special EquipmentWater baths preset to 42℃ and 95℃ and the appropriate annealing temperatureWhatman 3 MM filter paperMethod- Preparation of the oligonucleotide probe1. Prepare following reaction mixture & incubate at 37℃ for 1 hroligonucleotide primer (5-7 pmoles or 60 ng)1 uldistilled deionized H2O6.5 ul10X kinase buffer1.5 ulpolynucleotide kinase (~10 units)1 ul[?ortexing for 20 sec, centrifuge for 2 min-> transfer aqueous layer to a fresh sterile microfuge tube5. Chloroform extraction again.6. Add 55 ul of 3 M sodium acetate (pH 5.2) + 1 ml of EtOH.-> vortexing & store at -70℃ for at least 1 hr7. Centrifuge at maximum speed for 15 min at 4℃. Remove & discard radioactive sup.-> wash pellet with 70% EtOH, centrifuge again, remove & discard sup.-> dry the precipitate in the air,-> dissolve the precipitate in 500 ul of TE (pH 7.6)8. Counting specific activity of probe-> 2 ul of radiolabeled oligonucleotide primer + 10 ml of scintillation fluid-> count in a liquid scintillation counter-> calculate specific activity (assuming 80% recovery, ~ 2x106 cpm/pmole primer)- Hybridization & Extension of the Oligonucleotide Primer9. Mix 104~105 cpm (20-40 fmoles) of probe + 0.5~150 ug of RNA sample-> add 0.1 vol. of 3 M sodium acetate (pH 5.2) and 2.5 vol. of EtOH-> vortex & store at -70℃ for 1 hr-> centrifuge at 4℃ for 10 min at maximum speed.-> wash the pel master mix: thaw a 300 ul aliquot of primer extension mix on ice-> add 3 ul of 1 M dithiothreitol + reverse transcriptase (1-2 units/ul)+ 0.1 unit/ul of protein inhibitor of RNase-> mix gently by inverting tube several times, store on ice14. Remove tubes from water bath & collect the fluid in the base of tubes by brief centrifuge15. Add 24 ul of primer extension master mix to each tube-> mix gently & collect the fluid in the base of tubes by brief centrifuge16. Incubate the tubes at 42℃ (or 37℃) for 1 hr for primer extension reaction17. Terminate reaction by adding 200 ul of TE (pH 7.6)-> add 100 ul of equilibrated phenol (pH 8.0) + 100 ul of chloroform-> vortex for 20 sec, centrifuge at room temperature for 4 min-> transfer aqueous layer to a fresh sterile microfuge tube18. Add 50 ul of 10 M ammonium acetate + 700 ul of EtOH-> vortex vigorously & incubate tubes at -70℃ for at least 1 hr- Purification & analysis of the primer extension products19. Centrifuge at 4℃ for 10 min, remove sacrylamide gel22. After appropriate running, stop the electrophoresis (distance checking via tracking dyes)-> remove a glass plate from the gel-> cut off a corner of gel for orientation marking23. In case of thin gel (0.4 mm thickness), proceed to step 26.In case of thick gel (1.0 mm thickness), fix the gel in TCA-> transfer gel attached glass plate to a tray containing an excess of 10% TCA-> gently rock or rotate for 10 min at room temperature24. Pour off 10% TCA & replace it with an excess of 1% TCA-> gently rock or rotate for 5 min at room temperature25. Pour off 1% TCA & rinse briefly the fixed gel with distilled deionized H2O26. Transfer the gel to the Whatman 3 MM filter paper (1 cm larger than gel on all side)-> cut off a corner of filter paper according to gel orientation marking27. Remove glass plate & dry the gel on vacuum gel dryer at 60℃ for 1~1.5 hr28. Develop gel at -70℃ for 24 hr - 28 hr-> [?-32P]ATP로 lable한 경우 signal이 무척 강해서 -70℃보다 높은 온도 에서는signal이 broad하게 퍼지기 때문에 sharp.
    자연과학| 2014.09.13| 5페이지| 1,000원| 조회(144)
    태그 primer, extension, primer extension
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  • Northern blotting
    Northern blotting
    Northern Blotting(Transfer and Fixation of Denatured RNA to Membranes)Ⅰ IntroductionTransfer of denatured RNA to membranesTransfer of RNA from gels to nylon membranes at neutral or alkaline pH-Transfer to positively charged nylon membranes at alkaline pH-Transfer to uncharged nylon membranes at neutral pHⅡ MATERIALS주의사항: protocol에 이용되는 시약들은 DEPC-treated H2O를 사용해야 합니다.DEPC(diethyl pyrocarbonate )는 histidine을 선택적으로 alkylation시키는 물질입니다. 대부분의 RNase들의 active site에는 Histidine이 있고 따라서 DEPC는 효과적으로 RNAse를 inactivation시킬 수 있는 것입니다.Buffers and SolutionsAmmonium acetate(0.1 M) with 0.5 ㎍/ml ethidium bromide (step 13)Methylene blue solution= 0.02% (w/v) methylene blue (Sigma, 89% pure) in 0.3 M sodium acetate (pH5.5)Soaking solution= for charged membranes, use 0.01 N NaOH combined with 3 M NaCl= for uncharged membranes, use 0.05 N NaOH0.2 x SSC with 1% (w/v) SDS20 x SSCTransfer buffer= for alkaline transfers to charged membranes, use 0.01 N NaOH with 3 M NaCl= for neutral transfers to uncharged memample, fractionated through agarose, by soaking the gel in the appropriate soaking solutionespecially useful if the gel contains > 1% agarose or is >0.5 cm thick or if the RNA to be analyzed is > 2.5 kb in length1.1 for transfer to uncharged nylon membranesa. Rinse the gel with DEPC-treated waterb. Soak the gel for 20 minutes in 5 gel volumes of 0.05 N NaOHc. Transfer the gel into 10 gel volumes of 20X SSC for 40 minutesd. Without delay, proceed directly with Step 2 to transfer the partially hydrolyzed RNA to an uncharged nylon membrane by capillary action1.2 for transfer to charged nylon membranesa. Rinse the gel with DEPC-treated waterb. Soak the gel for 20 minutes in 5 gel volumes of 0.01 N NaOH/3 M NaClc. Without delay, proceed directly with Step 2 to transfer the partially hydrolyzed RNA to a positively charged nylon membrane by capillary action2. Move the gel containing fractionated RNA to a glass baking dish, and use a sharp scalpel to trim away unused areas of the gel. Cut alonill the dish with the appropriate transfer buffer (0.01 N NaOH/3 M NaCl for positively charged membranes, and 20X SSC for uncharged membranes) until the level of the liquid reaches almost to the top of the support.When the blotting paper on the top of the support is thoroughly wet, smooth out all air bubbles with a glass rod or pipette.Preparation of the Membrane for Transfer5. Use a fresh scalpel or a paper cutter to cut a piece of the appropriate nylon membrane ~1 mm larger than the gel in both dimensions6. Float the nylon membrane on the surface of a dish of deionized water untill it wets completely from beneath, and then immerse the membrane in 10X SSC for at least 5 minutes.Use a clean scalpel blade to cut a corner from the membrane to match the corner cut from the gel.Assembly of the Transfer system and Transfer of the RNA7. Carefully place the gel on the support in an inverted position so that it is centered on the wet blotting paper8. Surround, but do not cover, the gel with Sa, Smooth out any air bubbles with a glass rod.11. Cut of fold a stack of paper towels (5-8 cm) just smaller than the blotting papers. Place the towels on the blotting papers. Put a glass plate on top of the stack and weigh it down with a 400g weight12. Allow upward transfer of RNA to occur for no more than 4 hours in neutral transfer buffer and ~1 hour in alkaline transfer buffer13. Dismantle the capillary transfer system.Mark the positions of the slots on the membrane with a ballpoint pen through the gel. Transfer the membrane to a glass tray containing ~300 ml of 6X SSC at 23℃. Place the tray on a platform shaker and agitate the membrane very slowly for 5 minutes.(To assess the efficiency of transfer of RNA, rinse the gel briefly in several changes of water and then stain it for 45 minutes in a solution of ethidium bromide. Examine and photograph the stained gel under UV illumination)14. Remove the membrane from the 6X SSC and allow excess fluid to drain away.Lay the membrane, RNA sincharged nylon membraneType of MembraneMethod of FixationOrder of StepsPositively charged nylonalkaline transfer1. stain with methylen blue2. Proceed to prehybridizationUncharged nylon or positively charged nylon (nonalkaline transfer)UV irradiation1. Fix the RNA by UV irradiation2. Stain with methylene blue3. Proceed to prehybridizationUncharged nylon or positively charged nylon (nonalkaline transfer)baking in vacuum oven or microwave oven1. stain with methylene blue2. Bake the membrane3. Proceed to prehybridizationTo Fix By BAKINGa. Allow the membrane to dry in air and then bake for 2 hours between two pieces of blotting paper under vacuum at 80℃ in a vacuum ovenorb. Place the damp membrane on a dry piece of blotting paper and heat for 2-3 minutes at full power in a microwave oven (750-900 W)To Cross-Link By UV IRRADIATIONa. Place the damp, unstained membrane on a piece of dry blotting paper and irradiate at 254 nm for 1 minute 45 seconds at 1.5 J/cm2b. After irradiation, stain the m훈
    자연과학| 2014.09.13| 5페이지| 1,000원| 조회(115)
    태그 RNA, NORTHERN BLOTTING, Transfer of RNA
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  • mRNA isolation (Micro-FaskTrack kit)
    mRNA isolation (Micro-FaskTrack kit)
    Micro-FastTrackTM 2.0 mRNA Isolation Kit (Invitrogen)Introduction【 The Micro-FastTrack≒2.0 mRNA Isolation Kit allows isolation of polyA+ RNA directly from cells (1 x 102 - 5 x 106), tissue (10-200 mg), or total RNA (100-500 レg) in 2-3 hours using minimal equipment (a water bath, a microcentrifuge, and a syringe fit with an 18-21 gauge needle) without the need for ultracentrifugation or guanidinium lysis.【 A typical mammalian cell contains about 10-11 g of RNA of which 1-5% is polyA+ RNA. The remaining RNA is mostly rRNA (80-85% of total) and low-molecular weight RNAs such as tRNA (15-20% of total). To separate the heterogeneous population of mRNA from the majority of the RNA found in the cell, affinity binding to oligo(dT) cellulose is used. This method exploits the major characteristic of mRNA, polyadenylation, to obtain intact, pure mRNA.【 The procedure described below is designed to remove DNA and proteins from your sample and allow selective binding of mRNA under high salt conditioting mRNAMaterialsMethodグ. Preparing sample (tissue cultured cells)1. Wash the cells in +4‘C phosphate buffered saline (PBS) solution .2. Transfer the cells to a 15 ml sterile, conical centrifuge tube. Centrifuge the cells and resuspend the pellet in 1 ml of PBS.3. Transfer the cells to a sterile microcentrifuge tube. Centrifuge the cells and continue with Step 4, or flash freeze the pellet in liquid nitrogen and store at -70‘C.4. Resuspend the cell pellet in 1 ml of Micro-FastTrack≒ 2.0 Lysis Buffer to lyse the cells. If the pellet was frozen and does not thaw quickly, place the tube with the cells and buffer in a 45‘C water bath for 1-2 minutes. Vortex to completely resuspend the cells.5. Pass the lysate 2-4 times through a sterile plastic 1 cc syringe fitted with a 18-21 gauge needle. If the lysate is still viscous, continue to pass the solution through the syringe until it is no longer viscous (up to 10 times).ケ. Isolating mRNA1. Incubate the cell lysate produced in Preparing Samplple.2. Adjust the NaCl concentration of the lysate to 0.5 M final concentration by adding 63 レl of the 5 M NaCl stock solution to each 1 ml lysate. Mix thoroughly.3. Shear any remaining DNA by passing the lysate 3-4 times (or more as needed) through a sterile plastic 1 cc syringe fitted with an 18-21 gauge needle. This will yield a cleaner mRNA preparation.4. Remove a vial of oligo(dT) cellulose from the dessicator and add cell lysate or total RNA solution to the vial. Be sure to label the tube with the name of your sample.5. Seal the tube and allow the oligo(dT) cellulose to swell for 2 minutes.6. Rock the tube gently at room temperature for 15 to 20 minutes (using a rocking platform or a rotator). For yeast RNA, increase the rocking time to at least 1 hour. Rocking or rotating increases the efficiency of mRNA binding to oligo(dT) cellulose.7. Centrifuge oligo(dT) cellulose at 4,000 x g in a microcentrifuge for 5 minutes at room temperature. Remove the supernatant carefully from the rat Step 1 until the buffer is no longer cloudy (at least 2 more times). Use 1.3 ml of Binding Buffer per wash.3. Gently resuspend the resin in 0.3 ml of Binding Buffer and transfer the sample to a spin-column (inside the spin-column/microcentrifuge tube set). Centrifuge at 4,000 x g for 10 seconds at room temperature. Repeat this process as many times as necessary to transfer all of the oligo(dT) cellulose into the spin-column.4. Remove the spin-column from the microcentrifuge tube and discard the liquid inside the tube.5. Place the spin-column back into the tube and add 500 レl of Binding Buffer. Centrifuge at 4,000 x g for 10 seconds at room temperature. Read the OD260 of the flow-through.6. Repeat Step 5 (at least 3 times) until the OD260 of the flow-through is < 0.05.7. Add 200 レl of Low Salt Wash Buffer and gently resuspend the resin with a sterile pipette tip. Be careful not to damage the membrane of the spin-column as you will lose the resin (and your sample). Centrifuge at 4,000d 100 レl of Elution Buffer and mix the buffer into the cellulose bed with a sterile pipette tip.3. Centrifuge at 4,000 x g for 10 seconds at room temperature, but DO NOT decant the liquid. The mRNA is now in the eluate.4. Add a second 100 レl aliquot of Elution Buffer to the column, mix into the cellulose, and centrifuge again for 10 seconds. Steps 2 through 4 will elute the mRNA into the microcentrifuge tube.5. Remove the column from the tube. The tube should now contain 200 レl. Do Not Discard. This is your mRNA sample.6. Precipitate the mRNA with 10 レl of 2 mg/ml glycogen carrier (supplied), 30 レl of 2 M sodium acetate (supplied), and 600 レl of 200 proof (100%) ethanol. Freeze on dry ice until solid. The mRNA may be stored as an ethanol precipitate at -80‘C until ready for use. The mRNA will remain stable for several months in this condition.7. Thaw and centrifuge in a microcentrifuge at maximum speed (16,000 x g) for 15 minutes at +4‘C.8. Remove the ethanol. Centrifuge briefly and re.20
    자연과학| 2014.09.13| 3페이지| 1,000원| 조회(95)
    태그 mRNA, mRNA isolation, Micro-FaskTrack
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