Invitrogen topo ta cloning kit manual
Cloning and Sequencing your PCR Product.
Sequencing There are several facilities you can use.
Sequence quality varies from fair to poor.
Longamp and pminit are trademarks topo of New England Biolabs, Inc.This product is intended for research purposes only.After spinning, aspirate off the supernatant or carefully pour off and blot with a kimwipe.Preparing DNA, there are 2 ways to prepare your DNA for sequencing: minipreps and PCR.2) For the transformation, the instructions say to incubate on ice for 5-30 minutes.Let stand for 5 minutes and then turn on vacuum for 5 minutes.The easiest way to resuspend the pellet is to run the bottom of the tube along the rack until no clumps are visible in the solution.The cloning kit has 2 parts: 1) A cloning box containing the vector and salt solution which is stored at -20C and can be found in the enzyme freezer.2) A box containing the competent cells, SOC, and pUC18 control DNA that is stored at -80C and is in the bottom compartment of the freezer next invitrogen to the gel room.The topo vector has an overhanging 3'.We tried it once invitrogen and it took one month to get sequences that were topo so full of N's they were not useable.The PCR method is faster and easier but occasionally people have found that it does not work well on their construct.Most people use a total of 20ng.Invitrogen has changed the kit several times in the last year. Longer PCR products will manual need longer extension times.
Make sure they ARE properly aligned OR YOU will lose your sample.Trademarks, onetaq, Q5, cloning quick-load, NEB and NEW england biolabs are registered trademarks invitrogen of New England Biolabs, Inc.However, they don't do runs on Friday.For PCR products, 10-20ng/100bp of PCR product and.2pm of primer in a total of 12ul.To ensure a good density of colonies you can plate 50ul on one plate and 200ul on the other.Be careful manual not manual angry to contaminate it or you will contaminate everyone else's transformation.Place the lid on the base.The vector should not be able to self-ligate.(note: If you are not processing range them immediately, keep the cells at 4C and spin them down just before processing.Amp plates are made by the kitchen and are in the coldroom at the end of the lab.After eluting sample cover with microseal film for storage.Phusion is a registered trademark and property of Thermo Fisher Scientific.You should do 2 controls:1) vector alone.For PCR products, 20ng DNA/100bp of PCR product and.5ul of our 10ng/ul primer stock in a total of 12ul.Any lysate that hasn't gone through manual by now isn't cloning going. Do not wait much longer than 5 minutes before adding the N3 (neutralizing) buffer or you may irreversibly denature your DNA.
Cover with a microseal film for storage.
If you grow them longer the Amp may go off.
It's best to thaw it on your bench.
THE turbo strips should BE directly above invitrogen topo ta cloning kit manual THE qiaprep strips.
To elute your DNA, place the plexiglass block in the bottom of the manifold.
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