1.  50-100mL receptive cells, thawed at room temperature, after completely thawed (about 1 minute) gently dust several times to evenly suspend the cells.

2.  Add 5mL of the connecting solution (up to all can be added, as long as the volume does not exceed 1/10 of the volume of the receptive cells), gently mix, and leave for 5 minutes at room temperature.

3.  The carrier of our company uses commercial receptive cells without ice bath and heat shock, and can obtain enough converters by placing them at room temperature for 5 minutes, if self-made in the laboratory.

4.  When receiving cells or efficiency is low, it can be carried out according to standard procedures. Add 300-500mL LB or SOC medium (without antibiotics) and oscillate for 10 min at 180 rpm at 37℃.

5.  Take 200mL bacterial solution, coating the plate and culture overnight (if few transformogenes are expected, in order to obtain more clones, centrifuge at 4000rpm for 1min, absorb part of the supernatant, retain 100-150mL, lightly suspend the bacterial body, and take all bacterial solution coating plate.

Note:

(1)  Generally, the medium (balanced to room temperature in advance) can be directly added to the 1.5mL centrifuge tube of the receptive cells, covered with the centrifuge tube cap, and fixed horizontally in the oscillating incubator for oscillating culture resuscitation, without the need to transfer to tube culture for resuscitation.

(2)  Generally, when the commercial receptor cells do not exceed 2kb of inserted fragments, a sufficient number of inverters can be obtained in 10 minutes of resuscitation. If the self-made receptor cells are used in the laboratory or the inserted fragments are long and few inverters, the resuscitation time can be increased to 30-60 minutes to obtain more inverters.

The positive rate of this product is quite high, under normal circumstances, you can achieve what you see is what you get, as long as the growth of the colony is normal (not contaminated bacteria, the number of transformers is not too small), basically contains insertion. Therefore, when the inserted fragment does not exceed 2-3kb, 1-2 bacteria can be directly selected for sequencing without identification.

1.  The plasmid was extracted with the bacterial solution of the white colony cultured above. If the inserted fragment was large, the inserted plasmid could be directly identified by electrophoresis to see the size of the plasmid. The inserted fragment could also be released by EcoR I/Ecor V double enzyme digestion or cut with other appropriate enzymes, and the fragment size could be checked by agarose gel electrophoresis to determine whether the target fragment was contained.

2.  Select colonies for direct PCR detection (see Molecular cloning version 3 or consult us).

3.  Universal M13F/M13R primer sequencing to determine whether the target clone is present.

Ⅰ pTOPO-T Vector                                                    Ⅱ  pTOPO-T Vector polyclonal locus sequence

pTOPO-T Universal primer sequence：

M13F: TGTAAAACGACGGCCAGT

M13R: CAGGAAACAGCTATGAC