367pEC-pfu

Cloning of 367pEC-pfu

Purpose: Cloning of367pEC-pfu

Date: 2013-5-

Notebook:

Page:

Clone ID:367

Experimenters: Shanyi

Results

cloning

 PCR, use the following two pairs of primers and use 381 as template

  PCR1

  Pfu-Nde1-F1
  tatgattttagatgtggattac

  Pfu-Kpn1-R1
 (ctggcttaacattaaaaaatcc ggtac)
 gtaccggattttttaatgttaagccag

 PCR2

  Pfu-Nde1-F2
  tgattttagatgtggattac

  Pfu-Kpn1-R2
  ctggcttaacattaaaaaatcc g
  cggattttttaatgttaagccag

PCR:

PCR 1
Pfu-Nde1-F1 + Pfu-Kpn1-R1

TATG---pfu--- GGTAC

PCR 2
Pfu-Nde1-F2 + Pfu-Kpn1-R2
AC----pfu---- C

Mix two PCR reactions, denature and reannealat least 25%

Nde1
TATG----pfu--- GGTAC
   AC----pfu---- C

Run gel, cut out bands, put into the same tube (i.e., one tube), add DG buffer, heat at 65 oC until dissolve, purify with spin column, elute with 20 ul water.
Heat 95 oC for 10min, turn off PCR machine, cool slowly to room temp.

Add to purified NdeI+KpnI digested vector (at least 1:4 ratio, 1 vector + 4 Insert),ligate and transform

Colony Screening

Date: 2013

DIGEST WITH NdeI+KpnI+XbaI

send 367-1 to sequence, there are several mutations needed to fix

Fix mutations

 PCR

  use 367-1 as template and use the following four pairs of primers to fix the mutations

Pfu-Nde1-F1 + Pfu-fix-R3===>660bp

Pfu-fix-F3 + Pfu-fix-R1===>1000bp

Pfu-fix-F1 + Pfu-fix-R2===>360bp

Pfu-fix-R2 + Pfu-Kpn1-R1===>300bp

use phusion HS polymerase, 24cycles

then join four fragments by PCR :(use 0.15ul Phusion HS polymerase and 0.1ul Q5 hot start polymerase for each PCR reaction, extention time:2min30s, 25cycles)

PCR 1
Pfu-Nde1-F1 + Pfu-Kpn1-R1

TATG---pfu--- GGTAC

PCR 2
Pfu-Nde1-F2 + Pfu-Kpn1-R2
AC----pfu---- C

Mix two PCR reactions, denature and reannealat least 25%

Nde1           Kpn1
TATG----pfu--- GGTAC
AC----pfu---- C

Run gel, cut out bands, put into the same tube (i.e., one tube), add DG buffer, heat at 65 oC until dissolve, purify with spin column, elute with 30 ul water.
Heat 95 oC for 10min, turn off PCR machine, cool slowly to room temp.

Digest 1454pEC vector with Nde1+ Kpn1

Add PCR products to purified NdeI+KpnI digested vector (at least 1:4 ratio, 1 vector + 4 Insert),

ligate(5ul 10*T4 ligase buffer and 2ul T4ligase )and transform NEB TURBO

Colony Screening

Date: 2013

DIGEST WITH NdeI+KpnI+XbaI

Cloning of 367pEC-pfu

Purpose: Cloning of367pEC-pfu

Date: 2013-9-

Notebook:

Page:

Clone ID:367

Experimenters: Shanyi

Results

FIX MUTATIONS

 PCR

  use 367FIX-1 as template and use the following THREE pairs of primers to fix the mutations

Pfu-Nde1-F1 + Pfu-fix4-R

Pfu-fix4-F + Pfu-fix5-R

Pfu-fix5-F + Pfu-Kpn1-R1

use phusion HS polymerase, 24cycles

then join four fragments by PCR :(use 0.15ul Phusion HS polymerase and 0.1ul Q5 hot start polymerase for each PCR reaction, extention time:2min30s, 25cycles)

PCR 1
Pfu-Nde1-F1 + Pfu-Kpn1-R1

TATG---pfu--- GGTAC

PCR 2
Pfu-Nde1-F2 + Pfu-Kpn1-R2
AC----pfu---- C

Mix two PCR reactions, denature and reannealat least 25%

Nde1           Kpn1
TATG----pfu--- GGTAC
AC----pfu---- C

Run gel, cut out bands, put into the same tube (i.e., one tube), add DG buffer, heat at 65 oC until dissolve, purify with spin column, elute with 30 ul water.
Heat 95 oC for 10min, turn off PCR machine, cool slowly to room temp.

Digest V15PET30B +1454pEC vector with Nde1+ Kpn1

Add PCR products to purified NdeI+KpnI digested vector (at least 1:4 ratio, 1 vector + 4 Insert),

ligate(5ul 10*T4 ligase buffer and 2ul T4ligase )and transform NEB TURBO

Colony Screening

Date: 2013

DIGEST WITH NdeI+KpnI+XbaI

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