酵母单杂
1、启动子构在载体pLacZi上；
2、基因构在载体pB42AD上；
接头引物
LACZ-F：5`-ATCTGTCGACCTCGAG-3` 酶切XhoI
LACZ-R：5`-GAGCACATGCCTCGAG-3`
PB42AD-F：5`-TGCCTCTCCCGAATTC-3` 酶切EcoRI
PB42AD-R：5`-CGAGTCGGCCGAATTC-3`
测序引物
Pb42AD-F：ACCAGCCTCTTGCTGAGTGGAGATGCC
Pb42AD-R：GAAGTGTCAACAACGTATCTA
PLacZ2U-F：GAATTCGAGCTCGGTACCC
PLacZ2U-R：GGACCTAATGTATAAGGAAAG
3、菌株：EGY48；
4、转化步骤参照一般的酵母转化，共转到2缺平板上；
5、显色培养基：
X-gal (20 mg/ml in DMF)
将5-溴-4-氯-3-吲哚-β-半乳糖苷 (X-GAL；#8060-1)溶解在二甲基甲酰胺(DMF)中，–20°C避光保存。
10 X BU salts
Dissolve the following components in 1 L (total) of H2O:
70 g Na2HPO4? 7H2O
30 g NaH2PO4
调pH为7，高压灭菌，室温保存。
SD/Gal/Raf 培养基的配制（包含X-gal和BU salts，并且灭菌完降到55度以下再加）
Final concentration
To prepare 1 L of medium
β-Galactose
2%
50 ml of 40% stock 20g
Raffinose
1%
25 ml of 40% stock 10g
10 X BU salts
1 X
100 ml of 10 X stock
X-Gal
80 mg/L
4 ml of 20 mg/ml
YNB
6.7g
Agar
20g
SD-URA-TRP
0.72g
倒平板，室温下凝固。倒置平板，用塑料套筒套住，黑暗保存。在4°C下最多至两个月。
注意事项：
A、 当加入BU salts和X-Gal时培养基温度不得超过55°C ，如果超过55°C BU salts会沉淀，X-Gal会降解。
B、半乳糖必需是高纯度的，并且葡萄糖含量要小于0.01%；
C、调节培养基pH到7，因为这个值接近β-半乳糖苷酶反应的最适pH，能够为β-gal反应提供必需的磷酸盐。
Figure 1. Map and multiple cloning site (MCS) of pLacZi.
pLacZi is a yeast integration and reporter vector for use with the MATCHMAKER One-Hybrid System. This plasmid contains the bacterial lacZgene downstream of the minimal promoter of the yeast iso-1-cytochrome C gene (PCYC1). Target elements can be inserted into the MCS upstream of the PCYC1-lacZreporter. Without activation from a cis-regulatory element, lacZ expression is very low when the vector is integrated into the yeast genome. The yeast URA3gene is used as a selectable marker for integration into the nonfunctionaluralocus of the YM4271 host strain after linearizing the vector at the NcoI or ApaI site. pLacZi cannot replicate autonomously in yeast. This plasmid contains the ampicillin resistance gene (Amp) and the Col E1 origin for selection and propagation in E. coli. Unique restriction sites are in bold
Figure 2. pB42AD plasmid map and MCS sequence.
(pB42AD was originally published as pJG4-5 in Gyuris et al., 1993.) pB42AD expresses cDNAs or other coding sequences inserted into the unique ECO RI and Xho I sites as translational fusions to a cassette consisting of the SV40 nuclear localization sequence, the 88 residue B42 acidic activator (AD), and the hemagglutinin epitope tag. Fusion protein expression is under the control of the GAL1 inducible promoter. May be propagated and selected for in E. coliand yeast. The TRP1 transformation marker is used for selection in yeast. Unique sites are in bold. A pB42AD insert sequencing primer is included in the MATCHMAKER LexA Two-Hybrid System for sequencing inserts.
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