5) Purify the vector using on Qiagen column (e.g. Quiaquick PCR Purification Kit). 6) In a 1.5 mL microcentrifuge tube, add: - 46 µL of blunt-end digested plasmid.
Purification of blunt-ended plasmid and addition of T-overhang 1) 1- In a 1.5 mL microcentrifuge tube, digest 10µg of plasmid with either - a blunt-end restriction enzyme (e.g. EcoRV) [Preffered] or - a 5’ overhang-end restriction enzyme filled-in with Klenow polymerase Mix by gentle vortex and centrifuge briefly. 2) Incubate at 37°C for 1-2 hours. 3) Run 1-2 µL on gel to ensure complete digestion. 4) Add 2 µL of 0.5 M EDTA to the tube at the end of digestion. 5) Purify the vector using on Qiagen column (e.g. Quiaquick PCR Purification Kit) 6) In a 1.5 mL microcentrifuge tube, add: - 46 µL of blunt-end digested plasmid - 15 µL of 5x TdT Buffer - 7.5 µL of CoCl2 - 1.5 µL of 1 mM ddTTP - 5 µL of Terminal Transferase (25 U/µL) 7) Mix gently and centrifuge briefly and incubate 1.5 hours at 37°C. The use of ddTTP prevents extension of a T-tail at the end of the DNA molecule. This could not be achieved with dTTP.
Purification and storage of T-vector 1) Purify the T-tailed DNA solution either using column or bu Phenol/Chloroform/Ethanol precipitation. 2) In a 1.5 mL microcentrifuge tube, add: - 44µL of purified plasmid as above - 5 µL of 10X ligase buffer - 3 µL of T4 DNA Ligase (1 U weiss/µL) Mix well and centrifuge briefly. Incubate overnight at 16°C. This step is not absolutely required as T/A cloning will be efficient at 70% without any further purification. If the vector used does not allow blue/white screening after cloning, ligation/gel purification increases T/A cloning efficiency to 90%.
Molecular cloning 6
Molecular cloning 3) Run a 1% agarose gel in TAE at 5V.cm-1 4) Excise the band with a scalpel blade and gel purify it (QuiaQuick Gel Exctraction Kit) The vector molecules on which a single T or no T was added will either form concatemers or self-ligate. Their size will be different from that of linear T-vector that has a T at both extremities. 5) Quantify purify T-vector and aliquot it at 25-30 ng/µL. Usually, ligation are done with 50/60 ng of vector.
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incidentally, bolts on via 2 studs through the bulkhead, and two bolts onto the top cowl panel. You can use Transit reservoirs to feed the master cylinders.
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7) Resuspend in 1 volume of ice-cold 10% glycerol. Glycerol washes are meant for competent cells that will be frozen. 8) Spin the bacteria at 4°C for 10 minutes ...
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We are now, perhaps, coming to the single most important point in the preparation of a car for rallying. To get the best out of a car, whatever its power, it is ...
the wheel nuts the correct torque is 55 lb ft for both aluminium and steel wheels ... If a wheel does come off, the car will usually land on the brake back plate, and this ... In theory, at least, this new rule should stop some of the weight ..... 60
