Ua ntej ntawm tag nrho, EcoR1 thiab Xba1 tawm 5' overhangs thaum lawv zom DNA (raws li Nde1). Nyob rau hauv ob qho tib si qhov 3' kawg yog recessed uas yog vim li cas polyermase tuaj yeem ntxiv cov hauv paus. Thib ob, tag nrho cov polymerases tsuas tuaj yeem ntxiv cov hauv paus rau 3 'qhov kawg, yog liKlenow yuav ntxiv 4 lub hauv paus rau qhov rov qab 3 ' kawg ntawm ob qhov chaw los tsim qhov kawg.
Puas txwv cov enzymes tawm 5 phosphate?
Vectors thiab inserts digested los ntawm kev txwv enzymes muaj qhov tsim nyog hloov kho lub davhlau ya nyob twg (5 'phosphate thiab 3' hydroxyl), thaum tawg tawg tsim los ntawm Polymerase Chain Reaction (PCR) yuav tsis.
Puas blunt kawg yuav ligated?
Blunt-end ligation
Blunt kawg ligation tsis koom nrog lub hauv paus-pairing ntawm protruding xaus, yog li txhua qhov kawg nkaus yuav raug ligated rau lwm qhov kawg. Blunt kawg tuaj yeem tsim los ntawm kev txwv enzymes xws li SmaI thiab EcoRV.
Puas yog Taq polymerase tsim blunt kawg?
Yes… Taq Polymerase ntxiv A-Overhangs yog tias koj khaws cov kauj ruam kawg txuas ntxiv ntawm 72 dC ntawm PCR. Feem ntau siv rau TA Cloning.
Vim li cas siv Klenow fragment hauv DNA sequencing?
Cov Klenow fragment yog qhov muaj txiaj ntsig zoo rau kev tshawb fawb raws li cov haujlwm xws li: Kev sib xyaw ntawm ob-stranded DNA los ntawm cov qauv ib leeg . Filling in receded 3' end of DNA fragments to make 5' overhang blunt . -zom zaws tawm 3 'overhangs.
