GST分子量：
构建pGEX-KG-YFG重组质粒
1、分析所感兴趣的基因(your favorite GENE, YFG)
Primer Premier 5.0软件，分析YFG含有哪些酶切位点，注意是否与pGEX-KG载体的多克隆位点有重合
2、确定合适的双酶切位点
NEB网站(www.neb.com) Double Digest Finder软件，查找最佳双酶切组合(下表)

NEB双酶切图谱
BamHI	EcoRI	NEBuffer EcoRI + BSA at 37°C.	BamHI may exhibit star activity in this buffer.
	XbaI	NEBuffer 3 + BSA at 37°C.	At least one enzyme has < 100% activity in this buffer, so additional units of enzyme and/or longer incubation time may be necessary.
	NcoI	NEBuffer 3 + BSA at 37°C.
	SalI	NEBuffer 3 + BSA at 37°C
	XhoI	NEBuffer 3 + BSA at 37°C.
SmaI	XbaI	NEBuffer 4 + BSA at 25°C with SmaI, then add XbaI and raise temperature to 37°C.	At least one enzyme has < 100% activity in this buffer, so additional units of enzyme and/or longer incubation time may be necessary.
	NcoI	NEBuffer 4 at 25°C with SmaI, then add NcoI and raise temperature to 37°C.
	XhoI	NEBuffer 4 + BSA at 25°C with SmaI, then add XhoI and raise temperature to 37°C.
	SacI	NEBuffer 4 + BSA at 25°C with SmaI, then add SacI and raise temperature to 37°C.
	HindIII	NEBuffer 4 at 25°C with SmaI, then add HindIII and raise temperature to 37°C.	At least one enzyme has < 100% activity in this buffer, so additional units of enzyme and/or longer incubation time may be necessary.
EcoRI	NcoI	NEBuffer EcoRI at 37°C.
	SalI	NEBuffer EcoRI + BSA at 37°C.
	XhoI	NEBuffer EcoRI + BSA at 37°C.
	SacI	NEBuffer 1 + BSA at 37°C.	EcoRI may exhibit star activity in this buffer.
	HindIII	NEBuffer EcoRI at 37°C.
XbaI	NcoI	NEBuffer 2 + BSA at 37°C.
	SalI	NEBuffer 3 + BSA at 37°C.	At least one enzyme has < 100% activity in this buffer, so additional units of enzyme and/or longer incubation time may be necessary.
	XhoI	NEBuffer 2 + BSA at 37°C.
	SacI	NEBuffer 4 + BSA at 37°C.	This buffer is not supplied with either enzyme. At least one enzyme has < 100% activity in this buffer, so additional units of enzyme and/or longer incubation time may be necessary.
	HindIII	NEBuffer 2 + BSA at 37°C.
NcoI	SalI	NEBuffer 3 + BSA at 37°C.
	XhoI	NEBuffer 2 + BSA at 37°C.
	SacI	NEBuffer 1 + BSA at 37°C.
	HindIII	NEBuffer 2 at 37°C.
SalI	XhoI	NEBuffer 3 + BSA at 37°C.
XhoI	SacI	NEBuffer 1 + BSA at 37°C.	At least one enzyme has < 100% activity in this buffer, so additional units of enzyme and/or longer incubation time may be necessary.
	HindIII	NEBuffer 2 + BSA at 37°C.
SacI	HindIII	NEBuffer 2 + BSA at 37°C.	At least one enzyme has < 100% activity in this buffer, so additional units of enzyme and/or longer incubation time may be necessary.

对照YFG、载体多克隆位点，确定上、下游酶切位点
3、设计PCR上、下游引物
Primer Premier 5.0软件，设计PCR上、下游引物
酶切位点外最多含6个碱基
3’端不是A，最好是G或C，但是不推荐使用GC或CG结尾
3’端至少保证有10个碱基完全配对
得分(Rating)大于70
[注意]
上游引物：是否添加适当碱基，确保不打乱开放阅读框
下游引物：添加终止密码子(UAA、UAG、UGA)
4、引物合成及保存
合成：上海生工生物工程技术服务有限公司(Email：beijing@sangon.com，Tel：81767586);纯化方法：柱层析or聚丙烯酰胺凝胶电泳?;价格1.30/碱基
保存：贮存浓度：100pmol/μl(100μM)，工作浓度：10pmol/μl(10μM)，-20°C保存
5、PCR扩增YFG
模板：质粒10ng/μl 稀释少量 -20°C保存
引物：10pmol/μl(10μM) -20°C保存
Taq酶：NEB Quick-Load Taq 2×Master Mix 扩增片段小于2.0kb
反应体系(配制时置于冰上)

	25μl反应体系	50μl反应体系
模板	1μl	2μl
上游引物	1μl	2μl
下游引物	1μl	2μl
2×Master Mix	12.5μl	25μl
去离子H2O	9.5μl	19μl

反应条件
(1) 预变性 94°C 5 min
(2) 变性 94°C 30 s
(3) 退火 待定 30 s
(4) 延伸 72°C 待定
(5) 重复2-5 25-30个循环
(6) 补平缺口 72°C 10 min
(7) 暂存 10°C