DNA amplification method
A promoter sequence and target technology, applied in the field of DNA amplification, to achieve the effect of improving the effective utilization rate, improving the utilization rate, and avoiding purification
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Embodiment 1
[0045] Apply method 1 of the present invention (method based on transposon), amplify 5 picograms (pg) DNA
[0046] 1. Prepare before starting:
[0047] Primer synthesis: transposon mosaic ends and mosaic end complementary sequences
[0048] STT_Met1 (SEQ ID No: 3):
[0049] 5'-AATTAATACGACTCACTATAGGGAGATGTGTATAAGAGACAG-3'
[0050] STT_Met2 (SEQ ID No: 4): 5'-pCTGTCTCTTATACACATCT-3'
[0051] Anneal to form a double-stranded sequence containing the T7 promoter sequence: STT_ME (10pmol / ul):
[0052] STT_ME consists of 100pmol of STT_Met1 (dissolved in annealing buffer: 10mMTris-HCl (pH7.5), 1mM EDTA, 0.1mMNaCl) and 100pmol of STT_Met2 (dissolved in annealing buffer: 10mMTris-HCl (pH7.5), 1mM EDTA, 0.1mMNaCl), etc. Volume mixed annealing (94°C for 5 minutes, gradually cooling down to 25°C at 0.1°C per second). STT_ME (50pmol / ul) was diluted with water to 10pmol / ul.
[0053] 2. Experimental steps:
[0054] Assembly of transposons containing T7 promoter sequences:
[0055] S...
Embodiment 2
[0107] Using method 2 of the present invention (random primer-based method), 5 picograms (pg) of DNA were amplified.
[0108] Random primers introduce T7 promoter sequences.
[0109] phi29 DNA Polymerase: NEB:Catalog#:M0269.
[0110] NEBbuffer4: NEB:Catalog#:B7004
[0111] T7 sequence (SEQ ID No: 2) with random primers (phosphorothioate modification) at the end:
[0112] 5'-AATTAATACGACTCACTATAGGGNNNNsNsN-3' (where s stands for phosphorothioate modification.)
[0113] DNA (5pg): 3ul
[0114] NEBbuffer4: 2ul
[0115] T7 promoter sequence (500uM) with random primers (phosphorothioate modification) at the end: 1ul
[0116] 10mMdNTP: 1ul
[0117] 100XBSA: 0.4ul
[0118] ddH2O: 10.6ul
[0119] 95°C for 3min, immediately place on ice for 3min
[0120] When done add:
[0121] phi29 DNA Polymerase: 2ul
[0122] Total 20ul
[0123] React at a constant temperature of 30°C for 30 minutes, and at 65°C for 10 minutes, and place it on ice immedi...
Embodiment 3
[0147] The present embodiment is a comparative example:.
[0148] Using a method based on multiple strand displacement reactions, 5 picograms (pg) of DNA were amplified. phi29 DNA Polymerase (NEB, Catalog #: M0269S)
[0149] DNA (5pg): 3ul
[0150] phi29DNAPolymeraseReactionBuffer: 5ul
[0151] Phosphorothioate-modified 6-base random primer (500uM): 2.5ul
[0152] 10mMdNTP: 2.5ul
[0153] 100XBSA: 1ul
[0154] ddH2O: 33.5ul
[0155] 95°C for 3min, immediately place on ice for 3min
[0156] When done add:
[0157] phi29 DNA Polymerase: 2.5ul
[0158] Total 50ul
[0159] React at a constant temperature of 30°C for 16h.
[0160] The product was purified using Agencourt AMPure XP (Beckman Coulter, Inc) magnetic beads. The overview is as follows: add 1 times the volume of magnetic beads to the amplified product, place it at room temperature for 5 minutes, absorb it on a magnetic frame for 5 minutes, remove the supernatant, wash twice with 70% a...
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