User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/10/17: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
(8 intermediate revisions by the same user not shown)
Line 21: Line 21:


The backbone vector has BSMBI cut site within the hygromycine resistance gene. I was removed though [http://openwetware.org/wiki/User:Behzad_Damadzadeh/Notebook/PcTF_Subcloning_in_E-coli/2013/10/10 Site Directed Mutagenesis]
The backbone vector has BSMBI cut site within the hygromycine resistance gene. I was removed though [http://openwetware.org/wiki/User:Behzad_Damadzadeh/Notebook/PcTF_Subcloning_in_E-coli/2013/10/10 Site Directed Mutagenesis]
----
'''PCR Reactions'''
{| class="wikitable" border=1 cellpadding="7" cellspacing="0"
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''BD002'''
| align="center" style="background:#f0f0f0;"|'''HPk'''
| align="center" style="background:#f0f0f0;"|'''CMV'''
| align="center" style="background:#f0f0f0;"|'''Reaction Conditions'''
| align="center" style="background:#f0f0f0;"|'''Gel Picture'''
|-
|{| DNA Template (µL)||0.1||0.1||0.1 || rowspan="6" |
* 95°C, 3 min.
* [95°C, 30 sec.; '''45'''°C, 40 sec.; 72°C, '''6''' min.] x35
* 72°C, 3 min.
* 4°C, ∞
|rowspan="6" | [[Image:20131020 171748 (2).jpg |thumb|300px| Column 1 is the vector backbone size: ~4600 bps, column 2 is the amplified HPK promoter size~516bps and column 3 is amplified CMV size: 588bps ]]
|-
| Forward Promer (µL)||1||1||1
|-
| Reverse Primer (µL)||1||1||1
|-
| 2X GoTag (µL)||50||50||50
|-
| dH2O (µL)||47.9||47.9||47.9
|-
| Tolal (µL)||100||100||100
|}
*'''Extracting linearized plasmids with Sigma PCR DNA Purification Kit '''
{|class="wikitable" border=1 cellpadding="7" cellspacing="0"
| align="center" style="background:#f0f0f0;"|'''DNA Part'''
| align="center" style="background:#f0f0f0;"|'''260/280'''
| align="center" style="background:#f0f0f0;"|'''ng/µL'''
|-
| CMV||1.9||42
|-
| HPK||1.8||70
|-
| Backbone Plasmid||1.8||209
|}
*Dilute the purified PCR product to 20 fmol/μL
** Measure ng/μL of the purified sample.
** The volume of purified DNA (x) you will need to dilute in a final volume of 20 μL = length in bp ÷ measured ng/μL * 20 fmols/μL * 650 fg/fmol ÷ 1,000,000 fg/ng * 20 μL final volume
** Formula: x = length in bp ÷ measured ng/μL * 0.013 * 20
'''Backbone vector''' <math> X= (4600 / 209 ) * 0.013 * 20 = 5.72</math>
'''HPK''' <math> X= (2203 / 70 ) * 0.013 * 20 = 8.18</math>
'''CMV''' <math> X= (2275 / 42 ) * 0.013 * 20 = 14.08</math>
{| class="wikitable" border=1 cellpadding="7" cellspacing="0"
| align="center" style="background:#f0f0f0;"|'''Reaction'''
| align="center" style="background:#f0f0f0;"|'''1 (CMV)'''
| align="center" style="background:#f0f0f0;"|'''2 (CMV)'''
| align="center" style="background:#f0f0f0;"|'''3 (HPK)'''
| align="center" style="background:#f0f0f0;"|'''4 (HPK)'''
| align="center" style="background:#f0f0f0;"|'''Thermal Cycling Reaction Conditions'''
|-
| 20 fmol of each DNA part (µL)||1+1||1+1||1+1|| 1+1 || rowspan='6' |
* [45°C, 2 min.; 16°C 5 min.] x25
* 60°C, 10 min.
* 80°C, 20 min.
*4°C, ∞
|-
| 10x T4 ligase buffer (Promega) (µL)||1||1||1|| 1
|-
| T4 ligase (NEB)  (µL)||0.25||0.25||0.25||0.25
|-
| BSMBI (µL)||0.5||0.5||0.5||0.5
|-
| dH2O (µL)||6.25||6.25||6.25||6.25
|-
| Total (µL)||10||10||10||10
|}
* Bacterial transformation , Long transformation protocol
** Add total volume (10.0 μL) to 50 μL BL21 and DH5α, Incubate on ice for 1 min., heat shock at 42°C for exactly 45 sec., immediately place on ice for 1 min.  Add 800 μL sterile SOC medium. Grow with shaking at 37°C for 45 min. Pellet the cells for 3 min. at room temp. Resuspend the cells in 100 μL LB + AMP antibiotic. Plate cells on pre-warmed LB agar + AMP antibiotic. Grow overnight at 37°C.
== Result ==
<font color="red">'''100s of colonies on plates with DH5α , No colonies on plates with BL21.'''

Revision as of 13:59, 4 November 2013

PcTF Subcloning in E. coli <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

Golden Gate Assembly of BD002 to Replace HsvtkTATA with CMV and HPK Promoters

Desired Constructs:

BD003: 5XGAL4-Spacer-CMV-Kozak-AMCyan-AmCyan-NLS-Stop

BD004: 5XGAL4-Spacer-HPK-Kozak-AMCyan-AmCyan-NLS-Stop

Existing construct is: 5XGAL4-Spacer-HsvTK-Kozak-AMCyan-AmCyan-NLS-Stop in V0200 mammalian vector

Amplification of CMV and HPK from KAH187 and KAH184 respectively.

The backbone vector has BSMBI cut site within the hygromycine resistance gene. I was removed though Site Directed Mutagenesis

PCR Reactions

Part BD002 HPk CMV Reaction Conditions Gel Picture
DNA Template (µL) 0.1 0.1 0.1
  • 95°C, 3 min.
  • [95°C, 30 sec.; 45°C, 40 sec.; 72°C, 6 min.] x35
  • 72°C, 3 min.
  • 4°C, ∞
Column 1 is the vector backbone size: ~4600 bps, column 2 is the amplified HPK promoter size~516bps and column 3 is amplified CMV size: 588bps
Forward Promer (µL) 1 1 1
Reverse Primer (µL) 1 1 1
2X GoTag (µL) 50 50 50
dH2O (µL) 47.9 47.9 47.9
Tolal (µL) 100 100 100


  • Extracting linearized plasmids with Sigma PCR DNA Purification Kit


DNA Part 260/280 ng/µL
CMV 1.9 42
HPK 1.8 70
Backbone Plasmid 1.8 209


  • Dilute the purified PCR product to 20 fmol/μL
    • Measure ng/μL of the purified sample.
    • The volume of purified DNA (x) you will need to dilute in a final volume of 20 μL = length in bp ÷ measured ng/μL * 20 fmols/μL * 650 fg/fmol ÷ 1,000,000 fg/ng * 20 μL final volume
    • Formula: x = length in bp ÷ measured ng/μL * 0.013 * 20

Backbone vector [math]\displaystyle{ X= (4600 / 209 ) * 0.013 * 20 = 5.72 }[/math]

HPK [math]\displaystyle{ X= (2203 / 70 ) * 0.013 * 20 = 8.18 }[/math]

CMV [math]\displaystyle{ X= (2275 / 42 ) * 0.013 * 20 = 14.08 }[/math]

Reaction 1 (CMV) 2 (CMV) 3 (HPK) 4 (HPK) Thermal Cycling Reaction Conditions
20 fmol of each DNA part (µL) 1+1 1+1 1+1 1+1
  • [45°C, 2 min.; 16°C 5 min.] x25
  • 60°C, 10 min.
  • 80°C, 20 min.
  • 4°C, ∞
10x T4 ligase buffer (Promega) (µL) 1 1 1 1
T4 ligase (NEB) (µL) 0.25 0.25 0.25 0.25
BSMBI (µL) 0.5 0.5 0.5 0.5
dH2O (µL) 6.25 6.25 6.25 6.25
Total (µL) 10 10 10 10
  • Bacterial transformation , Long transformation protocol
    • Add total volume (10.0 μL) to 50 μL BL21 and DH5α, Incubate on ice for 1 min., heat shock at 42°C for exactly 45 sec., immediately place on ice for 1 min. Add 800 μL sterile SOC medium. Grow with shaking at 37°C for 45 min. Pellet the cells for 3 min. at room temp. Resuspend the cells in 100 μL LB + AMP antibiotic. Plate cells on pre-warmed LB agar + AMP antibiotic. Grow overnight at 37°C.

Result

100s of colonies on plates with DH5α , No colonies on plates with BL21.

Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Behzad_Damadzadeh/Notebook/PcTF_Subcloning_in_E-coli/2013/10/17&oldid=753689"