Efficient preparation kit for competent cells

Efficient preparation kit for competent cells

Catalog Number:30018

Composition, storage, and stability of the reagent kit:

Short term use at 4 ℃ to avoid pollution. Long term storage - valid for one year at 20 ℃.

The specific product parameters are subject to the parameters in the product manual received

Product Introduction

1. Efficient preparation kit for competent cellsIt is a modified version of the traditional efficient preparation method for sensory cells, with convenient operation and high conversion efficiency.

2. The use of this reagent kit can achieve a sensory efficiency of 10⁸Cfu/μ g plasmid. For small plasmids, the efficiency is slightly higher, while for large plasmids, the efficiency is slightly lower. The highly efficient competent cells prepared using this reagent kit can not only transform plasmids, but are also very suitable for transforming ordinary ligation products, especially for situations that require high transformation efficiency such as flat end ligation.

3. The use of this reagent kit is simple to operate, and it only takes about 60 minutes to prepare efficient competent cells after bacterial cultivation.

4. This kit is suitable for the vast majority of common Escherichia coli, including Top 10, DH5 α, BL21 (DE3), JM109, TG1, HB101, and XL-1. However, the conversion efficiency may vary greatly among different bacterial strains.

5. This reagent kit can be used multiple times, and a total of 100 μ l of competent cells can be prepared.

v Notes:

1. All reagents, consumables, instruments, and equipment must undergo sterilization treatment.

2. Antibiotic free LB is used in the preparation of competent cells. Antibiotic free LB must also be used during the 37 ℃ culture after heat shock when using competent bacteria, even if the transferred plasmid is resistant.

3. After the preparation of sensory cells is completed, please quickly transfer to -70 ℃.

Operation steps:（Please read the precautions before the experiment

1. Flat coating:

To achieve optimal sensory efficiency, glycerol bacteria or other forms of preserved bacterial strains must be coated on LB plates and cultured overnight.

2. Vaccination:

Take a LB plate with freshly cultured bacterial strains, and perform subsequent operations in a clean bench. Dip the tip of the tweezers in 70% alcohol and slightly burn it on an alcohol lamp to keep the tip of the tweezers sterile. Use tweezers to pick up a sterile plastic tip or toothpick, select a monoclonal antibody from a flat plate, and then place the plastic tip or toothpick dipped in the bacterial strain into a bacterial culture tube containing 3 milliliters of SOB or LB (customers should choose the appropriate culture medium according to the different bacterial strains). The above operation can also be carried out using an inoculation ring or the like.

3. Cultivation:

Cultivate overnight at around 250rpm at 37 ℃, and the optimal cultivation time is usually around 16 hours. It should never exceed 18 hours.

4. Re inoculation and cultivation:

According to the required amount of highly efficient competent cells, inoculate and culture with freshly cultured overnight bacteria at a ratio of 1:500. For example, take 100 microliters of fresh overnight bacteria and culture them in 50 milliliters of SOB or LB at 37 ℃ at approximately 250 rpm.

5. Preparation of sensitive cells:

ð Attention: The centrifuge speed and centrifugation time are very important for the production of competent cells. The optimal speed and centrifugation time for different types of bacteria vary and should be optimized according to specific situations. The optimal situation is to have an appropriate rotational speed and centrifugation time to allow the bacteria to centrifuge and settle, without being too tightly packed. Otherwise, it will be difficult to resuspend and can only be resuspended by forcefully blowing and hitting, which will cause damage to the cells and affect conversion efficiency.

1) When the OD600 of the cultured bacteria reaches around 0.5, place the cultured bacterial solution in an ice bath and cool for 15 minutes. Attention: All subsequent operations must be performed at 4 degrees Celsius or in an ice bath.

2) Centrifuge at 3000-3500g for 5 minutes at 4 ℃ (centrifuge must be pre cooled) to collect bacteria, discard the supernatant (try to remove the supernatant as much as possible, the less residue the better).

3) If the bacterial count before centrifugal precipitation is 50 milliliters, proceed with subsequent operations. If it is other volumes, convert them proportionally and proceed with subsequent operations.

4) Gently resuspend the bacterial precipitate in 10 milliliters of pre cooled and highly efficient solution A (to facilitate resuspension, a small amount of solution A can be added first, and then the remaining solution A can be resuspended). When blowing and resuspending, be sure to be very gentle, otherwise it will affect the effect.

5) Ice bath for 15 minutes.

6) Centrifuge 3000-3500g at 4 ℃ (centrifuge must be pre cooled) for 5-7 minutes to collect bacteria and discard the supernatant.

7) Gently resuspend the bacterial precipitate in solution B prepared with 2 milliliters of pre cooled high-efficiency sensory state. When blowing and striking heavy suspensions, be sure to be very gentle, otherwise it will affect the effect.

8) Ice bath for 15 minutes.

9) On an ice bath, it can be divided into 50-200 microliters per tube as needed.

10) Immediately use or freeze in liquid nitrogen or ethanol dry ice bath and store at -70 ℃.