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Kit Contents

Item #B25005    Size: 0.5 mg (blue cap)

Item #B25010    Size: 1.0 mg (violet cap)

Item #B25050    Size: 5 x 1.0 mg (violet cap)

Item #B25100    Size: 10 X 1.0 mg (violet cap)

Contains both of the following:

Item #P00100    1 mg CaCl2 dried (green cap)

Item #P00200    2.0 mL of nuclease-free H2O (clear cap)

Storage

Store at room temperature (dried or hydrated).

BAPCs with fluorescent dye must be stored in the dark.

Note: The critical step for transfecting successfully with this kit is determined by finding the best N/P ratio between the positive charges (N for Nitrogen) on the BAPtofect carrier and the negative charges (P for Phosphate) on the nucleic acid you are trying to deliver. Depending on the kit you received, #B25005 or #B25010, they will contain a total of 3.3 x 1017 or 6.6 x 1017 positive charges, respectively. The number of negative charges per 100 ng of nucleic acid will depend on whether it contains RNA or DNA. There are 1.8 X 1014 negative charges in 100 ng of RNA  and 1.7 x 1014 negative charges in 100 ng of DNA. Since you are working with weights, it does not matter if the nucleic acid is single or double stranded.

BAPtofect-25™ Procedure Details

Prepare Stock Solutions

  1. Dilute 0.5/1.0 mg of BAPC containing 3.3/6.6 x 1017 positive charges stock solution in 50 µL of water to yield 6.6 x 1015/ 1.3 x 1016 positively charged amino groups (N) per µL.
  2. Dilute CaCl2 with 1 mL nuclease-free H2O to yield 10 mM CaCl2 solution.
  3. Prepare nucleic acid solution as salt free solution in sterile water.
  4. Combine appropriate volume of BAPCs from 50 µL stock solution with salt free nucleic acid solution.
  5. See table below for sample volumes of BAPCs needed to create different N/P rations for 100 ng of nucleic acid. If you are using a different amount of a nucleic acid, you will need to adjust the volume of the BAPCs. This calculation can be made using a simple ratio equation: BAPC volume for a given N/P ratio divide by 100 ng = new BAPC volume/your nucleic acid nanogram amount. Most applications can achieve excellent transfection rates with N/P ratios of +2 to +20.
    1. Alternatively, BAPC aliquots can be dried prior to adding DNA solution to BAPC in order to achieve maximum concentration during BAPC / nucleic acid complexation
N/P µL BAPC ng of DNA expts per kit*
20 5/2.5 100 10/20
15 3.8/1.9 100 13/27
10 2.5/1.3 100 20/40
5 1.25/.65 100 40/80
2 0.5/.25 100 100/200
  1. Vortex mixture and allow at least 30 minutes for complex formation.
  2. Add 1 µL CaCl2 stock solution to the BAPC nucleic acid mixture.
  3. Incubate mixture for another 30-60 minutes.
  4. Add to cells and incubate for 4 hr, replace media and check after 48 hr.

Note:  Best results are seen with sub-confluent cultures. Since each cell type is different, experimenting with different ratios within this range is recommended.

Note:  Purchased nucleic acid solutions should be requested as “desalted”.  Nucleic acid molecules are highly charged and cations, such as sodium and magnesium ions, will neutralize the negative charges  and reduce the repulsive interactions between the phosphates.  High salt concentrations may result in BAPC / nucleic acid complex aggregation.