How much amount of recombinant plasmid DNA is required for DH5 alfa competent cell's transformation.
Dear Monika
1-For transformation, you could use very less DNA, 1 ng. You need to run a positive transformation control and calculate your transformation efficiency.
2-Information: You must use plasmid for calculating transformation efficiency. If you use a ligation product, your calculation will be incorrect due to the low amount of circularized DNA in a ligation reaction.
3-Calculate your transformation efficiency with the following equation (CFU is colony forming units): (# colonies on plate/ng of DNA plated) X 1000 ng/µg = CFU/µg of DNA
The measurement "ng of DNA plated" refers to how much DNA was plated onto each agar plate, not the total amount of DNA used per transformation. You can calculate this number using the following equation:
volume of plasmid used in µL x concentration of DNA in ng/µ x (volume plated / total reaction volume)
Example: You transformed 1µL of 0.05 ng/uL plasmid from the Transformation Efficiency Kit (note: 50 pg/µL = 0.05 ng/µL) into 100 µL of competent cells. You added 900 µL SOC to your cells to get a total reaction volume of 1000 µL and then plated 100 µLs of the transformation. The plate has 250 colonies on it the next day.
ng of DNA plated calculation: 1 µL x 0.05 ng/µL x (100 µL plated / 1000 µL total reaction volume) = 0.005 ng DNA plated
Efficiency calculation: (250 colonies / 0.005 ng) X 1000 ng/µg = 5 x 107 CFU/µg DNA
Please you can read the details of protocol at this link: http://2015.igem.org/Troubleshooting/Transformation
Good luck
Marius
Dear Monika,
5-10 ng of plasmid/cosmid DNA are required.
http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=1771
Hoping this will be helpful,
Rafik
Dear Monika
1-For transformation, you could use very less DNA, 1 ng. You need to run a positive transformation control and calculate your transformation efficiency.
2-Information: You must use plasmid for calculating transformation efficiency. If you use a ligation product, your calculation will be incorrect due to the low amount of circularized DNA in a ligation reaction.
3-Calculate your transformation efficiency with the following equation (CFU is colony forming units): (# colonies on plate/ng of DNA plated) X 1000 ng/µg = CFU/µg of DNA
The measurement "ng of DNA plated" refers to how much DNA was plated onto each agar plate, not the total amount of DNA used per transformation. You can calculate this number using the following equation:
volume of plasmid used in µL x concentration of DNA in ng/µ x (volume plated / total reaction volume)
Example: You transformed 1µL of 0.05 ng/uL plasmid from the Transformation Efficiency Kit (note: 50 pg/µL = 0.05 ng/µL) into 100 µL of competent cells. You added 900 µL SOC to your cells to get a total reaction volume of 1000 µL and then plated 100 µLs of the transformation. The plate has 250 colonies on it the next day.
ng of DNA plated calculation: 1 µL x 0.05 ng/µL x (100 µL plated / 1000 µL total reaction volume) = 0.005 ng DNA plated
Efficiency calculation: (250 colonies / 0.005 ng) X 1000 ng/µg = 5 x 107 CFU/µg DNA
Please you can read the details of protocol at this link: http://2015.igem.org/Troubleshooting/Transformation
Good luck
Marius
Hi there,
If the transformation is just about a purified plasmid to enter the bacterial cell, as little as 0.1ng DNA is enough as what you are looking for is at least one event of transformation (provided the cell prep is sufficiently competent). If the transformation is made with a ligation mix then you need more DNA (10s of ng) as the goal is to select the right construct among the transformants (so it's about the selection of a double event: the right ligation event followed by the successful transformation event).
Hi again,
If was you call "recombinant plasmid" is a purified plasmid and the purpose is to get isolated transformants on a selective plate and provided the recipient strain is competent enough, as little as 0.1ng in a transformation mix is enough.
Thank you liger. I wanna ask what are the ways to confirm that prepared competent cell is sufficiently competent?
When you get at least 100 cfu from a transformation made with 0.1ng of circular plasmid (ideally pUC18 or pUC19). This level of competency (106 cfu/µg of DNA) is the lower limit required for efficient cloning purpose.
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