拟南芥酵母文库构建.doc

拟南芥酵母文库构建

一．材料：
1．total RNA trizol 法提取 1mgRNA（两批材料）：
拟南芥 7天幼苗全株：250ug
14天幼苗全株：250ug
早期花序：已刚看见花蕾，未抽薹为准，250ug
晚期花序：已开花的花序，250ug

2．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) Primer
1. Combine the following reagents in a sterile 0.25-ml microcentrifuge tube:
3ul mRNA
1.0 ìl CDS III Primer
4.0 ìl Total volume
2. Mix contents and spin briefly.
3. Incubate at 72°C for 2 min.
4. Cool on ice for 2 min.
5. Spin briefly.
6. Add the following to the reaction tube:
2.0 ìl 5X First-Strand Buffer
1.0 ìl DTT (20 mM)
1.0 ìl dNTP Mix (10 mM )
1.0 ìl MMLV Reverse Transcriptase
9.0 ìl Total volume
7. Mix gently by tapping. Spin briefly.
8. Incubate at 42°C for 10 min.
9. Add 1.0 ìl 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 tube to room temperature, then add 1.0 ìl (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 at –20°C.
First-strand cDNA can be stored at –20°C for up to three months.
4. BD Matchmaker Library construction 试剂盒 ds cDNA 扩增：
Amplify ds cDNA by Long Distance PCR (LD-PCR)
1. Preheat the PCR thermal cycler to 95°C.
2.
2 ìl First-Strand cDNA
70 ìl Deionized H2O
10 ìl 10X BD Advantage 2 PCR Buffer
2 ìl 50X dNTP Mix
2 ìl 5' PCR Primer
2 ìl 3' PCR Primer
10 ìl 10X GC-Melt Solution
2 ìl 50X BD Advantage 2 Polymerase Mix
100 ìl Total volume
3. Mix gently by flicking 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 sec
68°C 6 minb
• 68°C 5 min
.
6. When the cycling is complete, analyze a 7-ìl aliquot of the PCR product from each sample
alongside 0.25 ìg of a 1-kb DNA size marker on a 1.2% agarose/EtBr gel. Typical results
obtained with Human Placenta Poly A+ RNA are shown in Appendix A. If your PCR product
does not appear as expected, refer to the Troubleshooting Guide.
7. Proceed with Section I or store ds cDNA at –20°C until use.
胶电泳图：

1 2 3 4 marker bp

2000

1000
750

500

250

100

1. 第一批样品ds cDNA 7ul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 2000marker

5. Purify ds cDNA with a BD CHROMA SPIN™ TE-400 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.
a. Prepare competent yeast cells (Appendix B).
b. In a sterile, prechilled, 15-ml tube combine the following:
• 20 ìl ds cDNA (from Section IX.I, Step 16)
• 6 ìl pGADT7-Rec (0.5 ìg/ìl)
• 20 ìl Herring Testes Carrier DNA, denatured*
*Transfer ~50 ìl 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 DNA
to the 15-ml reaction tube.
c. Add 600 ìl of competent cells to the DNA.
d. Gently mix by vortexing.
e. Add 2.5 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 ìl DMSO, mix, and then place the tube in a 42°C water bath for 20 min. Mix cells
every 10 min.
i. Centrifuge at 700 x g for 5 min.
j. Discard the supernatant and resuspend in 3 ml of YPD 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 (0.9%).
2. Select transformants on SD/–Leu plates.
a. Spread 200 ìl on each 150-mm plate (150 plates total).
Note: To check the transformation efficiency, spread 100 ìl of a 1:10, 1:100, 1:1,000, and 1:10,000 dilution on
100-mm SD/–Leu plates.
b. Incubate plates upside down at 30°C until colonies appear (~3–6 days).
c. Calculate the transformation efficiency.
results:

1:10, 616个
1:100, 66个
1:1,000, 10个
1:10,000 1个
》2.08 x 106 transformants / 3 ìg pGADT7-Rec

3. Harvest (pool) transformants.
a. Chill plates at 4°C for 3–4 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. Mix well.
e. Check the cell density using a hemacytometer. the cell density 》2 x 107 cells/ml, 。
f. Aliquot (1-ml) and store at –80°C.
g. To determine the library titer, spread 100 ìl of a 1:100, 1:1,000, and 1:10,000 dilution on
100-mm SD/–Leu plates. Incubate at 30°C until colonies appear (~2–3 days). Count the
colonies (cfu) and calculate the number of clones in your library.
Colonies:
11.46 x 107 个/ML,

4．PCR Colony Screening:
This procedure uses the BD Matchmaker GAL4 AD LD-Insert Screening Amplimer Set
(#9103-1) and BD Advantage™ 2 PCR Polymerase Mix. We recommend using the BD
Advantage 2 Polymerase Mix, rather than any other DNA polymerase formulation, because we
find that it performs well in yeast cell samples, and because it is optimized for applications that
involve longer templates and require high fidelity.

1. Preheat a PCR thermal cycler to 94°C.
2. Place the BD Advantage 2 PCR Kit components and GAL4 AD LD-Insert Screening
Amplimers on ice and allow them to thaw completely. Mix each component thoroughly
before use.
3. Prepare a Master Mix by combining the components as specified in Table XIII.
TABLE XIII: ASSEMBLING MASTER MIXES FOR PCR COLONY SCREENING
POLYMERASE MIX (Mg 2+ included in the buffer)
PCR-grade deionized H2O ：451 ìl
10X Advantage 2 PCR Buffer ：55 ìl
5' LD Amplimer (20 ìM： 11 ìl
3' LD Amplimer (20 ìM) ： 11 ìl
50X dNTP Mix (10 mM ea.) ： 11 ìl
50 X Advantage 2 Polymerase Mix ： 11 ìl
Total ：550 ìl l
4. Using a sterile pipette tip, scrape a few cells from a colony that you wish to analyze. Place
the cells in the bottom of a clean 250-ìl PCR tube.
5. Add 24 ìl of Master Mix to the tube. Gently pipette up and down to disperse the cells.
6. Cap the tube and place it in a preheated thermal cycler.
7. Begin thermal cycling，use the following program:

• 94°C 3 min
• 25–30 cycles:
94°C 30 sec
68°C 3 min
• 68°C 3 min
• Soak at 15°C
8. Analyze the PCR product 。

Rapid cloning by homologous recombination is extremely efficient and reliable. After cotransforming yeast with pGADT7-Rec (Sma I-linearized) and cDNA (prepared with the SMART method outlined in the text), transformants were selected on SD/–Leu plates. Randomly picked clones were screened for inserts by PCR. In this analysis, 23 out of 23 transformants carried plasmids with cDNA inserts. Note the wide range of insert sizes.

bp Marker 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

2000
1000
750
500
250
100