拟南芥酵母文库构建

1、拟南芥酵母文库构建一材料：1total RNA trizol法提取 1mgRNA(两批材料)：拟南芥 7 天幼苗全株： 250ug14 天幼苗全株： 250ug早期花序：已刚看见 xx，未抽薹为准， 250ug晚期花序：已开花的花序， 250ug2 total RNA 1mg经 promega mRNA isolation试剂盒纯化沉淀浓缩成 10ul。3 BD Matchmaker Library construction 试剂盒第一链合成 :Synthesize First-Strand cDNA using an Oligo (dT) Primer1. Combine the follo

2、wing reagents in a sterile0.25-ml microcentrifuge tube:3ul mRNA1.0 l CDS III Primer4.0 l Total volume2. Mix contents and spin briefly.3. Incubate at 72 C for 2 min.4. Cool on ice for 2 min.1/ 95. Spin briefly.6. Add the following to the reaction tube:2.0 l 5X First-Strand Buffer1.0 l DTT (20 mM)1.0

3、l dNTP Mix (10 mM )1.0 l MMLV Reverse Transcriptase9.0 l Total volume7. Mix gently by tapping. Spin briefly.8. Incubate at 42 C for 10 min.9. Add1.0 l BD SMART III Oligonucleotide.10. Incubate at 42 C for 1 hr.11. Place the tube at 75 C for 10 min to terminate first-strand synthesis.12. Cool the tub

4、e to room temperature, then add1.0 l (2 units) RNase H.13. Incubate at 37 C for 20 min.15. Any first-strand reaction mixture that is not used right away should be placed at20C.First-strand cDNA can be stored at20 C for up to three months.4.BD Matchmaker Library construction 试剂盒 ds cDNA扩增：Amplify ds

5、cDNA by Long Distance PCR (LD-PCR)1. Preheat the PCR thermal cycler to 952/ 9C.2.2 l First-Strand cDNA70 l Deionized H2O10 l 10X BD Advantage 2 PCR Buffer2 l 50X dNTP Mix2 l 5 PCR Primer2 l 3 PCR Primer10 l 10X GC-Melt Solution2 l 50X BD Advantage 2 Polymerase Mix100 l Total volume3. Mix gently by f

6、licking the tube. Centrifuge briefly.4. Cap the tube and place it in a preheated (95 C) thermal cycler.5. Begin thermal cycling. If you have a hot-lid thermal cycler, use the following program:? 95 C 30 sec?20 cyclesa:95 C 10 sec68 C 6 minb? 68 C 5 min.0.25 g of a 1-kb DNA size marker on a3/ 91.2% a

7、garose/EtBr gel. Typical resultsobtained with Human Placenta Poly A+RNA are shown in AppendixA. If your PCR productdoes not appear as expected, refer to the Troubleshooting Guide.7. Proceed with Section I or store ds cDNA at20 C until use.胶电泳图：1 2 3 4 marker bp200010007505002501001.第一批样品 ds cDNA 7ul

8、 上样2. 第一批样品第一链 cDNA 1ul 上样3. 第二批样品 ds cDNA 7ul 上样4. 第二批样品第一链 cDNA 1ul 上样5.0.1ug 2000marker5. Purify ds cDNA with a BD CHROMA SPIN? -T4E00 Column，结合两批材料样 品沉淀浓缩成 40ul。二 Constructing & Screening a Two-Hybrid Library按 Protocol A 构建：1. Transform yeast strain AH109 with ds cDNA and pGADT7-Rec.4/ 9? 20 l d

9、s cDNA (from Section IX.I, Step 16)? 6 l pGADT7-Rec (0.5 g/ l)? 20 l Herring Testes Carrier DNA, denatured*Transfer 50 l of Herring DNA to a microcentrifuge tube and heat at 100 C for 5 min. Then,immediately chill the DNA by placing the tube in an ice bath. Repeat once more before adding the DNAto t

10、he 15-ml reaction tube.A.d. Gently mix by vortexing.e. Add2.5 ml PEG/LiAc Solution.f. Gently mix by vortexing.g. Incubate at 30 C for 45 min. Mix cells every 15 min.h. Add 160 l DMSO, mix, and then place the tube in a 42 C water bath for 20 min. Mix cellsevery 10 min.i. Centrifuge at 700 x g for 5 m

11、in.j. Discard the supernatant and resuspend in 3 ml ofYPD Plus Liquid Medium.k. Incubate at 30 C with shaking for 90 min.l. Centrifuge at 700 x g for 5 min.m. Discard the supernatant and resuspend in 30 ml of NaCl Solution (5/ 90.9%).2.Select transformants on SD/Leu plates.a. Spread 200 l on each 15

12、0-mm plate (150 plates total).Note:To check the transformation efficiency, spread 100 l of a 1:10, 1:100, 1:1,000, and 1:10,000 dilution on100-mm SD/Leu plates.b. Incubate plates upside down at 30 C until colonies appear (36 days).c. Calculate the transformation efficiency.results:1:10,616 个1:100,66

13、个1:1,000,10个1:10,0001 个 2.08 x 106 transformants / 3 g pGADT7-Rec3. Harvest (pool) transformants.a. Chill plates at 4 C for 34 hr.b. Add 10 ml Freezing Medium to each plate.c. Use sterile glass beads and gentle swirling to dislodge the cells into the liquid.d. Combine all liquids in a sterile flask.

14、 Mix well.e. Check the cell density using a hemacytometer. the cell density 2 x 10 7cells/ml,。f. Aliquot (1-ml) and store at 80C.6/ 9g. To determine the library titer, spread 100 l of a 1:100, 1:1,000, and 1:10,000 dilution on100-mm SD/ Leu plates. Incubate at 30 C until colonies appear (23 days). C

15、ount thecolonies (cfu) and calculate the number of clones in your library.Colonies:11.46 x 107 个 /ML,4 PCR Colony Screening:find that it performs well in yeast cell samples, and because it is optimized for applicationsthatinvolve longer templates and require high fidelity.1. Preheat a PCR thermal cy

16、cler to 94C.TABLE XIII:ASSEMBLING MASTER MIXES FOR PCR COLONY SCREENINGPOLYMERASE MIX (Mg2+included in the buffer)PCR-grade deionized H2O： 451 l10X Advantage 2 PCR Buffe：r 55 l5 LD Amplimer (20 M： 11 l3 LD Amplimer (20 M)：11 l7/ 950X dNTP Mix (10 mM ea.：) 11 l50 X Advantage 2 Polymerase Mi：x 11 lTotal：550 l l4. Using a sterile pipette tip, scrape a few cells from a colony that you wish to analyze. Placethe cells in the bottom of a clean 250- l PCR tube.5. Add 24 l of Master Mix to the tube. Gently pipette up and down to disperse the cells.6. Cap the tube and pla