JPH01157388A - Transformer method - Google Patents

Abstract

PURPOSE:To prevent deterioration of receptor cell of freezer storage, by thawing a receptor cell in a plastic type throwaway test tube, carrying out transforming treatment of the thawed cell and applying heat pulse required for the transforming treatment to the test tube using a heat source of high-temperature solid. CONSTITUTION:A receptor cell 2 of Escherichia coli. bacterium previously treated with calcium chloride is poured into a throwaway test tube 1 several times at an amount of e.g. about 100ml per time and frozen and stored. When transforming treatment is carried out, receptor cell 2 of the above-mentioned plastic throwing test tube is thawed and then transforming treatment is carried out and heat pulse required for the transforming treatment is applied to the test tube 1 by a heat source of high-temperature solid. To be concrete, the above-mentioned treatment is carried out by inserting the test tube 1 into a bored hole 4 of heated block 3 made of aluminum and retaining the heating for prescribed time.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to a method for transforming E. coli.

[Conventional technology]

A common method for transforming E. coli is to use recipient cells treated with calcium chloride. At this time, the recipient cells conventionally prepared prior to transformation are cryopreserved in a vial, thawed when performing the transformation operation, and the amount to be used (approximately α1 to α2s+j per time) is The remaining recipient cells are then re-frozen and preserved in a test tube.

In the transformation operation, the DNA suspension was mixed with recipient cells and kept at 0°C, and then 57 to 4
A rapid heating to 5° C. is carried out. This is called heat pulse application, and is an important operation in transformation.

[Problem that the invention seeks to solve]

In the conventional method, the recipient cells are stored and repeatedly thawed and frozen each time a transformation operation is performed, and the transformation efficiency of the recipient cells tends to gradually decrease. Since the unsealing and dispensing operations are repeated, the operations are complicated, and contamination by various bacteria during the process also becomes a problem.

Therefore, the present inventor thought of dispensing and storing recipient cells in small-capacity disposable test tubes with plastic lids in single-use amounts, and then performing the transformation operation in these test tubes. When heat pulses are applied according to the conventional heating method, the glass test tube wall has a higher heat transfer resistance than the glass test tube wall, so the recipient cells inside cannot be heated quickly and the transformation cannot occur. Not only is the efficiency significantly reduced, but the small external size of small-volume glass test tubes, which have a good shelf life, makes them susceptible to contamination of the internal receptor cell sample by the water in the water bath when heated in a water bath. A problem arose in that it was more susceptible to bacterial contamination than the law.

[Purpose of the invention]

It is an object of the present invention to provide a method by which the above-mentioned transformation reaction can be carried out in a light test tube using a plastic test tube.

[Problem t-Means for solving]

That is, in the present invention, prior to the transformation operation, calcium chloride 7
The E. coli recipient cells treated with E. coli are aliquoted into usable amounts in disposable plastic test tubes and stored frozen, and when performing the transformation procedure, the plastic cleft disposable test tubes are prepared. The transformation process is carried out after the recipient cells in the tube are thawed, and the heat pulse necessary for the transformation process is applied to the plastic test tube using a high-temperature solid heat source.

In the present invention, for example, recipient cells can be prepared for one use, t
o o pt was aseptically dispensed into single-use glass test tubes, stored frozen, thawed before use, and the subsequent transformation operations were performed in these single-use test tubes. By performing the important heat pulse application using a solid heat source with a temperature higher than the target heating temperature, the conventional problems are solved and the operation is simplified.

[Effect]

Since only the stored recipient cells that will be used are taken out and thawed, there is extremely little risk that the remaining recipient cells will deteriorate. Furthermore, since the number of devices that the sample comes into contact with during the transformation operation and the number of operating steps can be reduced, bacterial contamination can be suppressed and the complexity of the operation can also be avoided.

Furthermore, by bringing a heat source higher than the heating target temperature of 37 to 45 degrees Celsius into contact with the plastic test tube, the heat transfer rate is increased and the heat transfer resistance is greater than that of the glass test tube, which is compensated for and the interoceptive cells are improved. Rapid heating and the use of a solid heat source in place of the traditionally used water bath eliminate water contamination of the recipient cell material.

[Example] Escherichia coli HB10 prepared according to the conventional calcium chloride method
A suspension of 16% glycerol and 1M calcium chloride of one strain of recipient cells was dispensed in 100 μt portions into a sterilized plastic test W (L5-volume) with an outer diameter of 11 days φ.
Stored frozen at ℃. This state is shown in FIG. In FIG. 1, 1 indicates a disposable test tube made of polypropylene, and 2 indicates a recipient cell.

Take one of the frozen test tubes, thaw it in water, and add 5μr/ml of plasmid DNA PBR322 to this recipient cell solution.
Add 2μ of the wt suspension and let stand at 0°C for 1 hour.

Next, as shown in Fig. 2, the polypropylene disposable test tube 1 containing this sample was inserted into the perforation 4 with an inner diameter of 1 t5 mφ in an aluminum block 6 kept at 68°C, and held for 2 minutes. did. During this time, the temperature inside test tube 1 rose to 42°C.

After this, 800 μt of the 2XYT liquid medium shown in Table 1 was added, and after culturing at 37°C for about 50 minutes, transformants were obtained according to a conventional method using the antibiotic ampicillin as a selection marker, and the transformation efficiency was examined. Plasmid PBR
322 DNA 1μ? There were 1×10′ transformants per cell. This is the same as the recipient cells immediately created by the conventional method.

Table 1 EACTol' Lipton (manufactured by DIFCO) 161DAC! To yeast extract (
(manufactured by DIpco) 101 Sodium Chloride
5? Water 1 was further prepared using the above method and stored frozen for 3 months, and the transformation efficiency in pnematoceptive cells was examined. There was no difference in the transformation efficiency with the pnematoceptive cells immediately after preparation, indicating that the recipient aF@ was stored stably for a long period of time. This was confirmed.

Also, no bacterial contamination was detected during this period.

〔Effect of the invention〕

According to the present invention, deterioration of cryopreserved recipient cells and bacterial contamination can be suppressed, and operations can be simplified.

[Brief explanation of the drawing]

Figure 1 is a fruit of the present invention! An explanatory diagram showing the caps of recipient cells cryopreserved in Gerastec test tubes as an ll1i row, and FIG. 2 shows the state of heating of a plastic test tube using an aluminum block as an example of the present invention. FIG.

Claims (1)

[Claims] Prior to the transformation operation, E. coli recipient cells that have been treated with calcium chloride are aliquoted into single-use plastic test tubes and stored frozen. Transformation is performed after thawing recipient cells in a disposable plastic test tube, and a heat pulse necessary for the transformation is applied to the plastic test tube using a high-temperature solid heat source. Conversion method.