Method for keeping activity of active protein under normal temperature
A technology of active protein and protein, applied in the biological field, can solve the problems of inconvenient storage and use of active protein, difficulty in long-term storage, inactivation of active protein, etc.
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Embodiment 1
[0062] Embodiment 1 Preparation of active protein protection composition of the present invention
[0063] 1. Preparation of storage solution
[0064] Preparation of 25% lauryl polyethylene glycol ether (Brij-35) stock solution: Weigh 25 g of Brij-35 (Calbiochem Company, item number: 203724-11) and dissolve it in about 80 ml of distilled water, heat to aid dissolution, and cool Add distilled water to 100ml. This stock solution is stable for 1 year at room temperature.
[0065] Preparation of 50% trehalose stock solution: Weigh 25 grams of trehalose (Sigma company, product number: T-5251), dissolve it in 40 milliliters of distilled water, and dilute to 50 milliliters. This stock solution is stable for 1 year at 4°C.
[0066] 2. Preparation of Active Protein Protection Preparation
[0067] The effective final concentration of Brij-35 is 0.1%, and the effective final concentration of trehalose is 5%. Preparation of Brij-35 / trehalose 10-fold stock solution: Take 24 ml of the ...
Embodiment 2
[0068] Embodiment 2 Restriction endonuclease test
[0069] Select 25 kinds of restriction endonucleases such as EcoRI, EcoRV, BamHI (see Figure 2), T4 DNA ligase, MMLV reverse transcriptase, Taq DNA synthetase and other modification enzymes as samples for testing. A detailed list of the above-mentioned various test samples is shown in Table 1. All enzymes are MBI products. The specific method is: take 200 μl of the above test sample enzyme, put it in a 1.5ml plastic centrifuge tube (eppendorf), add 20.0 μl of the above 10× storage solution, mix well and divide it into three 200 μl PCR tubes, each tube 20 μl. Use the same conditions but without adding Brij-35 / trehalose as negative control.
[0070] The specific test is as follows:
[0071] (1) Put each of the above enzymes at 42°C for treatment, 3 tubes for each temperature. The maximum processing time is 730 days (2 years). In the meantime, 1 μl was taken from each of the 7th, 14th, 21st, 28th, 70th, 365th, and 730th day...
Embodiment 3
[0078] Example 3 DNA ligase test
[0079] The pUC57 vector (purchased from MBI) was digested with EcoRI / HindII, and the gel was recovered after digestion. The insert fragment is a fragment of about 500 bp recovered from pA336 (purchased from MBI, pUC57 with a 3kb insert fragment amplified from lambda) plasmid after EcoRI / HindII double digestion. The connection was carried out according to the manufacturer's instructions, and the blue and white spots were screened. Count the number of white plaques in 1000 clones. The results are shown in Table 1.
[0080] Table 1
[0081] Processing time (days)
25℃
37℃
65℃
7
227
278
205
321
8
14
229
229
281
245
1
21
303
247
256
281
0
28
256
238
241
303
0
70
277
...
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