主题：【原创】这是真的吗？Maxipreps concentration＝4163.1ng/ul？！ -- 大鹏翔宇
我一个天才的高中同学现在就在费城搞这玩意呢。
原理看明白了，不过，如果city1和2自己就互补上了咋办？有什么办法避免吗？
Computer Made from DNA and Enzymes:
A year ago, researchers from the Weizmann Institute of Science in Rehovot, Israel, unveiled a programmable molecular computing machine composed of enzymes and DNA molecules instead of silicon microchips. Now the team has gone one step further. In the new device, the single DNA molecule that provides the computer with the input data also provides all the necessary fuel.
当时我提出了个设想，以DNA氢键断裂能量为计算媒介代替0和1，以蛋白质为输出方式的并行式生物计算机。
不知莲兄可否看过刘慈新的《天使时代》，与这个思路正好相反。
Nice figure.
I have several questions/suggestions, though.
1. It would be more informative if you could label the DNA ladder, I tried to guees the size but I could not correlate the sizes.
2. You may want to try to load less sample. It looks to me that you've loaded more than you need - or may be it's just the result of photographying. As a matter of fact, you might be able to distinguish the two bands in lane 5/9 if you chose carefully the amount of sample loaded and the gel concentration.
3. Load the empty lane with loading buffer (prefereably the same ionic strenght as the samples) might help to straighten the running lanes.
4. Lane 9 shows you have not completely digested the sample. The tails in Lane 7-9 may indicate that you have some impurities in your phRL-SV40 vector preparation. This might explain partially why you got the high yield - you could get some E coli genome DNA with your plamid. However, this is just my guessing.
There is a step after you broke the E coli where you need mix that gelly stuff with buffer. You need to mix them gentlely, otherwise, you might break some genome DNA, which will not be separated with centrifugation since it will not precipitate. In another word, it will go with your plasmid sample.
[The above are just my own opinion. We can discuss them if you have different thoughts.]
这是一个1kb ladder，不可否认没有分开，其实这个电泳跑得并不好，因为做大胶的设备被打包了（要搬家），只能用小胶但小胶板只能做8个大槽，显然不够，所以做了15个小槽。
样品的确是加多了，所以看起来有点模糊，从上之下依次为：10kb，9kb，8kb，7kb，6kb，5kb＋4kb（连在一起，就是紧贴着2上面的那一条），3kb，2kb，1kb，0.5kb。
sample7－9跑歪了，因为跑胶一开始的时候盛胶的塑料盘有点歪。您的方法我下次试试。
您说的不纯导致的浓度偏高可能是对的，是不是需要再分离一次呢？
Is your vectors empty or you have already put something in it (1.8 - 2k bps?)? It looks to me that the size of DNA fragments in gel are larger than what it should be based on the plamid map.
Usually I would not bother to do it if it's a vector. Reason: 1. If you will use it as stock, only plasmid can transform when you want to amplify it; 2. If you want to use it for ligation, you will purify the fragment after the digestion, which will separate them. However, it may consume some of your enzyme, you need take it into account.
对不起，这一段我看得不明白，您是指哪一段呢？
Take lane 3 for example: the plasmid is 4045 bp according to the map while the DNA fragment in lane 3 is somewhere around 6k bp. That's why I said you have already put something (a gene) in the vector.
I think you should have since you will use it for transfection. Are you going to use luciferase as a reporter gene?
至于那个6kb的条带，可能是contamination，也可能是digested linear plasmid。
我是要用luciferase assay的，刚刚做完，我的第一次luciferase assay。
多少毫升菌液？曾用500毫升菌液提得4毫克多质粒，还是20KB左右的。所以和商家的最高提取量没多大关系。不过现在都用Gerard Biotech的midi kit,50ml菌液基本上能提1毫克，最多的一次100ml提了5毫克还多，不过用这个试剂盒得从Promega另外定resin去除内毒素，总的说来很不错的一牌子，优点是价格出奇地低。
看来小兄弟手艺不错，是干这些活的料子，前途光明
最近实验室空调坏了，所以俺天天都在33度的高温中做实验，maxipreps是做不成了，以后要学着做midiprep了，至于价格，您猜得没错，俺们老板是个老抠门