基因克隆2014 version by Ailing Lu1，载体的选择：常用的载体有两种：一是克隆载体，另外一种是表达载体，看自己的需要选择载体。
2，酶切位点的选择.在载体的多克隆位点选择酶切位点，单酶切或双酶切都可，尽量选择双酶切。
并且这些酶切位点在特异性的ＰＣＲ产物中不可存在。
在设计PCR引物时在特异性的ＰＣＲ序列两端加上所选的双酶切位点，酶切位点外加上适量的保护碱基３，PCR扩增对于PCR扩增其实不同的基因可能策略，所选的扩增酶的也不同，有高保真的，也有保真性不高的。
但是如果用表达载体，设计引物时必须考虑开放阅读筐，保证表达蛋白的不移码突变。
第一次先做小体积PCR检测PCR条件是否合适，如果合适，做100ul PCR反映体系。
４，PCR产物的酶切,一般ＰＣＲ产物需要纯化，纯化后进行PCR产物酶切。
５，质粒的酶切.（1）、虽然质粒的酶切很简单但是却很讲究,决定着克隆的成败.质粒提取我一般都用试剂盒,质粒提取完毕后我会用紫外分光光度法对质粒进行定量测定,根据A260的值计算出质粒的量,然后再进行酶切。
做一般的克隆酶切２ug载体，胶回收，足够做克隆。
酶的用量为：1ug DNA, 3-5U 的限制性内切酶足够，不可多用，以避免星形活性。
酶切中加入1%的BSA可以提高酶切效果酶切总体一般为酶切2-4小时足够。
（2）、双酶切如果两种酶所用缓冲液成分不同（主要是盐离子浓度不同）或反应温度不同，这时可以采用如下措施解决：1）先用一种酶切，然后乙醇沉淀回收DNA分子后再用另外一种酶切；2）先进行低盐要求的酶酶切，然后添加盐离子浓度到高盐的酶反应要求，加入第二种酶进行酶切；3）使用通用缓冲液（通用缓冲液查询冰箱门上，或网上查询，网站neb）进行双酶切。
具体要根据酶的反应要求进行，尽量避免星号活力。
（3）、对提取质粒做酶切鉴定时，400ng的质粒用于酶切，3U的限制性内切酶，0.2 ul 的BSA, 20ul的酶切体系，37℃, 酶切1-2h就可以了。
（4）、酶切后的基因需要用PCR纯化试剂盒纯化后做连接实验。
酶切注意事项：（1）.酶切的选择原则一般是尽量扩大酶切体系，这样抑制因素得以稀释；基因组DNA或质粒DNA酶的用量较一般DNA大，一般为1μg/10U；所加酶的体积不能超过酶切总体积的1/10，否则甘油浓度会超过5%，会产生星号活力；对难切的质粒或基因组DNA应延长反应时间4―5hr，甚至过夜。
灭活限制性内切酶活性可以采用加热灭活，乙醇沉淀，酚/氯仿抽提，添加EDTA或SDS等方法，具体每一种酶可能有些方法不能完全灭活，这一点需要注意。
（2）.双酶切体系缓冲液添加量要根据两种限制性内切酶最佳缓冲体系，可以登录限制性内切酶购买公司官方查看。
（3）.在酶切反应缓冲液中加入BSA，通过提高溶液中蛋白质的浓度，对酶起保护作用。
6，连接连接体系中质粒载体的质量要求达到25ng，XY实验时，用到70ng。
For purified PCR amplicon products, the amount of insert to be added can be calculated by relative length or molar calculations. Formulas below use the recommended values of 25 ng of linearized vector per reaction and an insert-to-vector ratio of 3:1-6:1For example:vector is 10kb, insert is 1kb,25ng10kb = 1/3, to calculate how much insert for ligationxng1kbAssemble the cloning reaction by：Vector Xul (25ng)T4 DNA ligase buffer 1 μlinsert Y μlNuclease-free H2O 5 μlT4 DNA ligase 1 μl.10ul1, Incubate at room temperature (25°C) for 15 minutes or at 16C for 2hour to overnight.A negative control cloning reaction should be performed with the linearized vector alone and no insert.2, Incubate on ice for 2 minutes, ready for transformation7, transformation: take 2-10ul for transformation, the left can be stored at -20C, for later transformation.连接产物和感受细胞的比例不能超过1：10.Gene cloning2014 version by Ailing Lu1, choose the vectorFor regular cloning, there are two kind of vectors, one is cloning vector, another one is expressing vector. Which vector you will use depend on your experiment.2, choose the cloning sitesChoose the cloning sites from vectors’MSC, single or double enzyme enzyme can,but first choice to choose the double enzyme digestion.Appropriate restriction sites, absent in the target gene, are incorporated in the forward and reverse primers when a target gene is generated by PCR. Add some bases at the end of cleavage.3, Primer Design and Cloning Hints:Design your PCR primers to include a sufficient overlap with the sequence of the gene you want to amplify.You may also want to include a stop codon at the C-Terminus of the fusion (in front of the downstream cloning site) in order to terminate translation at that position.For fusions upstream of the MCP-tag, ensure that a start codon is included. The addition of a Kozak sequence (e.g. GCCRCCATG, where the start codon is underlined) will increase the translation efficiency.In general, any linker peptide between the proteins should be kept short to avoid degradation by proteases. If required, specific protease cleavage sites can be introduced into the linker peptide. Care should be taken to design the cloning so that the fusion partners in the resulting construct are in frame.Perform the PCR reaction and subsequent cloning steps according to established protocols for molecular biology.After subcloning the gene of interest into pMCP-tag(m) Vector as a fusion with the MCP gene, the resulting plasmid can be used for transient expression of the MCP-tag fusion proteins in a suitable cell line.Direct cloning can also be used to make fusions with the MCP-tag. This is only possible if the fusion partner has compatible sites adjacent to the gene of interest.Care should be taken to design the cloning so that the fusion partners in the resulting construct are in frame.4，PCR amplification, usually choose the high-fidelity taqpolymerase to do PCR. First time to test the PCR condition, if the PCR works well, do 100ul PCR for purification5，Digest purified PCR product and vector plasmid, usually 2ug vector for digest is enough Remember to use 3-5U restrict enzyme for 1ug DNA, digest for 2-4 hours, don’t use more restrict enzyme.6，LigationFor purified PCR amplicon products, the amount of insert to be added can be calculated by relative length or molar calculations. Formulas below use the recommended values of 25 ng of linearized vector per reaction and an insert-to-vector ratio of 3:1-6:1For example:vector is 10kb, insert is 1kb,25ng10kb = 1/3, to calculate how much insert for ligationxng1kbAssemble the cloning reaction by：Vector Xul (25ng)T4 DNA ligase buffer 1 μlinsert Y μlNuclease-free H2O 5 μlT4 DNA ligase 1 μl.10ul1, Incubate at room temperature (25°C) for 15 minutes or at 16C for 2hour to overnight.A negative control cloning reaction should be performed with the linearized vector alone and no insert.2, Incubate on ice for 2 minutes, ready for transformation7, transformation: take 2-10ul for transformation, the left can be stored at -20C, for later transformation.。
