The volume of water = 20 µl - 5 µl - 1 µl (for Ligase enzyme) - 2 µl (volume of pBluescript) - 0.83 µl (volume of Lambda DNA) = 11. Since the total volume is 20 µl, the volume of water required will be. The melting temperature (Tm) is defined as the temperature at which half of the DNA strands are in the random coilor single-stranded (ssDNA) state. Therefore, the volume of Lambda DNA required is 0.83 µl. Nucleic acid thermodynamicsis the study of how temperatureaffects the nucleic acid structureof double-stranded DNA(dsDNA). Volume of Lambda DNA = Amount of Lambda DNA / Concentration of Lambda DNA ![]() Since the concentration of Lambda DNA is 8 ng/µl, the volume of Lambda DNA can be calculated as. To achieve a 1:3 (vector: insert) ratio of DNA, the amount of Lambda DNA required to mix with the pBluescript DNA is required to be calculated-įirst, Calculation of the amount of pBluescript DNA is required -Īmount of pBluescript DNA = 2 µl (volume) x 10 ng/µl (concentration of pBluescript DNA) = 20 ngĪmount of Lambda DNA = 1/3 x Amount of pBluescript DNA = 1/3 x 2 x 10 ng/µl ![]() It is commonly used as a cloning vector due to the presence of multiple cloning site (MCS) regions with the compatibility of several restriction enzymes. PBluescript vectors are the cloning vectors that contain circular double-stranded (ds) DNA (deoxyribonucleic acid) plasmid.
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