DNA molecules are connected to each other

T4 phage DNA ligase differs from E. coli DNA ligase in that it catalyzes the ligation of blunt-ended DNA fragments (Sgaramella and Khorana, 1972; Sgaramella and Ehrlich, 1978). This is an extremely useful enzyme because DNA is easily blunt-ended. Learn to be physical. With such physical properties, any DNA molecules can be connected to each other.

advantage:

1. If you can't connect, you can't open it. The blunt-end ligation does not involve the problem of base sticking at the sticky end, so theoretically any two DNA sequences can be joined together, of course requiring the enzymatic action of ligase. The connection between the different DNA molecules is greatly facilitated because the blunt ends are naturally so, and if they encounter a 5' or 3' protruding sticky end, they can also be treated as blunt ends.

2. Sometimes, after the two blunt ends are joined together, another endonuclease recognition sequence can be generated, providing an opportunity for subsequent cloning work.

Disadvantages:

1, the connection efficiency is lower than the sticky end: the reason why the connection efficiency is relatively low, one of the reasons is that only the role of ligase in the process of ligation, lack of sticky ends to highlight the complementary role of the base; the second reason is the commonly used T4DNAligase for the flat The Km value at the end is 1000 times higher than the sticky end.

2, may produce two-way insertion: because the end of the flat end connection is not specific, the connection can not be oriented, so the insertion in both directions is possible. This time you need an effective identification method. Generally, an enzyme cleavage site is selected for the restriction enzyme digestion in the vector and the target fragment. Of course, the specific identification method is determined according to the specific experiment.

3. Multiple copy insertions may be possible: When the flat end is connected, there may be multiple insertions of the target segments, and it is also possible that each of the inserts is connected in different directions at the time of multiple insertions, which sometimes makes the results difficult to interpret. The larger the general purpose segment, the smaller the possibility of multiple copy insertion.

Flat end connection requirements:

1. Low concentration (0.5 mmol/L) of ATP (Ferretti and Sgaranekka, 1981).


2. There is no polyamine such as spermidine.

3. Very high concentration of ligase (50 Weiss units. ml).

4, high concentration of flat end.

Oven Thermometer

For cooking purposes- to measure the temperature inside an oven-, you need a thermometer with special characteristics. First, you need something that has a much higher temperature range than your typical household thermometer. Best oven thermometers typically have a range that goes from 50 to 300 degrees Celsius or 100 to 550 degrees Fahrenheit.

The mechanism that makes an oven thermometer work is also different from traditional thermometers. Instead of infrared sensors of mercury, they have a strip made of two pressed-together metals that expand and contract at different temperatures. This movement makes the dials of the face move to the right temperature indicator. Easy, right? Now to choose the best and most accurate one.

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