User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/05/24: Difference between revisions

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* Add C-terminal His<sub>8</sub>-tag after CBD.
* Add C-terminal His<sub>8</sub>-tag after CBD.


==Materials==
==Special Materials==
===[http://www.openbiosystems.com/Query/?i=0&q=EBT3996-99602816, BSA clone]===
===[http://www.openbiosystems.com/Query/?i=0&q=EBT3996-99602816, BSA clone]===
* BSA cDNA in pCMV-SPORT6
* BSA cDNA in pCMV-SPORT6
Line 55: Line 55:
## Miniprep - Thurs
## Miniprep - Thurs
# Dilute primers to 100 μM with sterile H<sub>2</sub>O
# Dilute primers to 100 μM with sterile H<sub>2</sub>O
## Dilute BSA cloning primers to 4 μM (?) for subcloning PCR
## Dilute BSA cloning primers to 4 μM for subcloning PCR
## Dilute His-tag primers to 125 ng/μL for QuikChange
## Dilute His-tag primers to 12.5 ng/μL for QuikChange
# BSA PCR - Thurs afternoon
# BSA PCR - Thurs afternoon
{|border="1"
|+
Basic PCR protocol
|-
!Tube !!sterile H<sub>2</sub>O !!Pfu Buffer !!Template !!5'primer !!3'primer !!dNTPs !!Pfu Turbo !!wax
|-
! !! !!(10X) !!(10 μg/mL) !!(4 μM) !!(4 μM) !!(10 mM ea) !!(2.5 U/μL) !!
|-
!A
|72 μL ||10 μL ||5 μL ||5 μL ||5 μL ||2 μL ||1 μL ||50 μL
|-
!B
|73 μL ||10 μL ||5 μL ||5 μL ||5 μL ||2 μL ||0 μL ||50 μL
|}
** Add each from left to right. Mix and spin before adding Pfu Turbo. Gentle mixing and spin after Pfu Turbo. Don't mix in wax.
## 5' @ 95°C
## 30" @ 95°C
## 2' @ 60°C
## 2' @ 78°C
## repeat steps 2-4 x 29 more times
## 5' @ 78°C
# analytical minigel - Fri afternoon
# purify by spin column - Fri afternoon
# QuikChange of pTXB1 (when plasmid arrives)
{|border="1"
|+
Basic QuikChange protocol
|-
!Tube !!sterile H<sub>2</sub>O !!Pfu Buffer !!Template !!For primer !!Rev primer !!dNTPs !!Pfu Turbo !!wax
|-
! !! !!(10X) !!(10 μg/mL) !!(12.5 ng/μL) !!(12.5 ng/μL) !!(10 mM ea) !!(2.5 U/μL) !!
|-
!A
|12 μL ||10 μL ||10 μL ||10 μL ||5 μL ||2 μL ||1 μL ||50 μL
|-
!B
|13 μL ||10 μL ||10 μL ||10 μL ||5 μL ||2 μL ||0 μL ||50 μL
|}
** Add each from left to right. Mix and spin before adding Pfu Turbo. Gentle mixing and spin after Pfu Turbo. Don't mix in wax.
## 30" @ 95°C
## 30" @ 95°C
## 1' @ 55°C
## 8.5' @ 68°C
## repeat steps 2-4 x 17 more times
## 10' @ 68°C
# DpnI digestion
## add 1μL DpnI to PCR tube
## 1h @ 37°C
# analytical minigel of pTXB1 PCR
# transform into DH10B
:* May need to make competent cells
# Miniprep
# sequence
# Sequential double-digest
## NheI digest
{|border="1"
|+
NheI digest protocol
|-
!Tube !!sterile H<sub>2</sub>O !!10X NEB2 !!100X BSA !!DNA !!NheI (10 kU/mL)
|-
!plasmid
|34.5 μL ||5 μL ||0.5 μL ||5 μL of 100-500 μg/mL ||5μL
|-
!insert
|24.5 μL ||5 μL ||0.5 μL ||15 μL from purified PCR reaction ||5μL
|}
### 2h @ 37°C
### 20' @ 65°C
## SapI digest
{|border="1"
|+
SapI digest protocol
|-
!Tube !!sterile H<sub>2</sub>O !!10X NEB4 !!DNA !!SapI (2 kU/mL)
|-
!plasmid
|30 μL ||5 μL ||10 μL of NheI digest ||5μL
|-
!insert
|30 μL ||5 μL ||10 μL of NheI digest ||5μL
|}
### 2h @ 37°C
### spin-column purify
## Phosphatase vector
## gel purify
## ligate
## transform DH10B
## miniprep
## sequence
## express


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|}

Revision as of 13:09, 24 May 2011

AU CHEM-570 Lab Prep <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

Objective

Subclone BSA into pTXB1

  • Use NheI and SapI cloning sites. This will fuse the BSA to a C-terminal intein and chitin binding domain (CBD).
  • Add C-terminal His8-tag after CBD.

Special Materials

BSA clone

  • BSA cDNA in pCMV-SPORT6
  • IMAGE clone ID # 8838824
  • E. coli DH10B TonA
  • glycerol stock frozen at -80°C

PCR primers

  • All primers from IDT
    • 100 nmol scale
    • HPLC purified
  • BSA cloning
    • BSA-NheI-5'
5'- CCC ACA AAA CTA TGG CTA GCA AGT GGG TGA CT-3'
Tm = 64.4°C
MW = 9842.4 g/mol
19.3 nmol, 0.19 mg
  • BSA-SapI-3'
5'-TGG TTG GCT CTT CTA CGG GCT AAG GCT GT-3'
Tm = 66.0°C
MW = 8946.8 g/mol
16.9 nmol, 0.15 mg
  • His-tag cloning
    • ins_His_after_CBD
5'-CCT TGT GGC AGC TTC AAC ACC ACC ATC ATC ATC ACC ACC ACT GAC TGC AGG AAG GG-3'
Tm = 72.1°C
MW = 17083.1 g/mol
8.0 nmol, 0.14 mg
  • Rins_His_after_CBD
5'-CCC TTC CTG CAG TCA GTG GTG GTG ATG ATG ATG GTG GTG TTG AAG CTG CCA CAA GG-3'
Tm = 72.1°C
MW = 17398.3 g/mol
7.4 nmol, 0.13 mg

pTXB1

  • From NEB
  • 200 μg/mL

Experimental Plan

  1. Miniprep BSA clone
    1. streak clone on LB+Amp100 plates - today
      1. O/N @ 37°C
    2. inoculate 5 mL LB+Amp100 - tomorrow
      1. O/N @ 37°C
    3. Miniprep - Thurs
  2. Dilute primers to 100 μM with sterile H2O
    1. Dilute BSA cloning primers to 4 μM for subcloning PCR
    2. Dilute His-tag primers to 12.5 ng/μL for QuikChange
  3. BSA PCR - Thurs afternoon
Basic PCR protocol
Tube sterile H2O Pfu Buffer Template 5'primer 3'primer dNTPs Pfu Turbo wax
(10X) (10 μg/mL) (4 μM) (4 μM) (10 mM ea) (2.5 U/μL)
A 72 μL 10 μL 5 μL 5 μL 5 μL 2 μL 1 μL 50 μL
B 73 μL 10 μL 5 μL 5 μL 5 μL 2 μL 0 μL 50 μL
    • Add each from left to right. Mix and spin before adding Pfu Turbo. Gentle mixing and spin after Pfu Turbo. Don't mix in wax.
    1. 5' @ 95°C
    2. 30" @ 95°C
    3. 2' @ 60°C
    4. 2' @ 78°C
    5. repeat steps 2-4 x 29 more times
    6. 5' @ 78°C
  2. analytical minigel - Fri afternoon
  3. purify by spin column - Fri afternoon
  4. QuikChange of pTXB1 (when plasmid arrives)
Basic QuikChange protocol
Tube sterile H2O Pfu Buffer Template For primer Rev primer dNTPs Pfu Turbo wax
(10X) (10 μg/mL) (12.5 ng/μL) (12.5 ng/μL) (10 mM ea) (2.5 U/μL)
A 12 μL 10 μL 10 μL 10 μL 5 μL 2 μL 1 μL 50 μL
B 13 μL 10 μL 10 μL 10 μL 5 μL 2 μL 0 μL 50 μL
    • Add each from left to right. Mix and spin before adding Pfu Turbo. Gentle mixing and spin after Pfu Turbo. Don't mix in wax.
    1. 30" @ 95°C
    2. 30" @ 95°C
    3. 1' @ 55°C
    4. 8.5' @ 68°C
    5. repeat steps 2-4 x 17 more times
    6. 10' @ 68°C
  2. DpnI digestion
    1. add 1μL DpnI to PCR tube
    2. 1h @ 37°C
  3. analytical minigel of pTXB1 PCR
  4. transform into DH10B
  • May need to make competent cells
  1. Miniprep
  2. sequence
  3. Sequential double-digest
    1. NheI digest
NheI digest protocol
Tube sterile H2O 10X NEB2 100X BSA DNA NheI (10 kU/mL)
plasmid 34.5 μL 5 μL 0.5 μL 5 μL of 100-500 μg/mL 5μL
insert 24.5 μL 5 μL 0.5 μL 15 μL from purified PCR reaction 5μL
      1. 2h @ 37°C
      2. 20' @ 65°C
    2. SapI digest
SapI digest protocol
Tube sterile H2O 10X NEB4 DNA SapI (2 kU/mL)
plasmid 30 μL 5 μL 10 μL of NheI digest 5μL
insert 30 μL 5 μL 10 μL of NheI digest 5μL
      1. 2h @ 37°C
      2. spin-column purify
    2. Phosphatase vector
    3. gel purify
    4. ligate
    5. transform DH10B
    6. miniprep
    7. sequence
    8. express


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