老师从小就教导我们:“先把数据算算对,再去做实验!还有,说的就是你啦,别忘了单位!”。不知道长做分子生物实验的同学们有没有这样的感觉,计算其实是一个十分重要的过程,这关系到整个实验的成功与否。有时候要算个摩尔数啊,再来个摩尔比啊,最后再来个单位换算什么的,一堆一堆,绝对不难。
切入正题,到底哪些公式会比较常用呢?小编认为有一下几种:
1. 摩尔浓度(mol/L)与溶质质量(g)
m (g) = Desired Molarity (mol/L) x V溶剂 (L) x M溶质 (g/mol)
2. 稀释倍数n 和稀释液的体积Vadding
n =(Cfinal * Vfinal)/(Cstock * Vstock)
Vadding = Vfinal – Vstock
Final=最终溶液
Stock=储备液/原液
3. 论OD260的重要性(当实验室的Nanodrop坏掉了或被坏人持续占用了)
CDNA/RNA (μg/ml) = Absorbance (OD260) * conversion factor
conversion factors:
- 1 OD260 Unit = 50 μg/ml, dsDNA
- 1 OD260 Unit = 40 μg/ml, ssRNA
- 1 OD260 Unit = 33 μg/ml, ssDNA
这里的μg/ml也就是ng/μl,所以和Nanodrop测出来的单位是相同的。值得注意的是,这里的每一个OD260­单位对应的核酸浓度只是估算值,毕竟碱基组成不同也会造成折光度的差异,所以在有的网站上,这三组值也会略有差异。
4. DNA/RNA 质量到摩尔数的估测转换
dsDNA:
molesdsDNA (mol) = mdsDNA (g)/[(length of dsDNA (bp) x 617.96 g/mol) + 36.04 g/mol]
ssDNA:
molesssDNA (mol) = mssDNA (g)/[(length of ssDNA (nt) x 308.97 g/mol) + 18.02 g/mol]
ssRNA:
molesssRNA (mol) = mssRNA (g)/[(length of ssRNA (nt) x 321.47 g/mol) + 18.02 g/mol]
这里bp就是碱基对数,nt就是核苷酸数。
正常估算来讲一对碱基的平均摩尔质量是650 g/mol,小编这里用的是NEB (New England Biolad)推出的修正公式。617.96 g/mol 其实是除去了每对碱基两端的水分子(聚合成链的过程是要除去一个H和一个OH的)。最后又加上36.04 g/mol,是假设两个-OH和两个-H分别加在最后大分子的两端。这样的算法比直接使用650 g/mol要稍微准确一点,但是整个过程还是建立在估算碱基对平均分子量的基础之上,所以不论哪种方法计算出来的都是平均值而已。
5. 粘合反应(ligation)插入片段和质粒的摩尔比例
Massinsert (g) = (insert/vector molar ratio) x massvector (g) x (ratio of insert/vector lengths)
一般这个插入片段对质粒的摩尔比例可以是2:1或者是3:1,都可以根据自己的实验而定。另外一个重要的比例就是插入片段对质粒的序列长度比。
6. 单位换算
1 ng = 10-3 μg =10-6 mg = 10-9 g = 10-12 kg
1 cm3 = 10-3 dm3 = 10-6 m3
1 cm3 = 1 mL
1 dm3 = 1 L
等等等……
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