Haynes Lab:Notebook/Engineering PC-TFs/2015/03/23: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
(Autocreate 2015/03/23 Entry for Haynes_Lab:Notebook/Engineering_PC-TFs)
 
Line 7: Line 7:
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
==Summary==
==Summary==
*  
* LCR
* Met Kodibagkar grad student Shubanghi
 
'''LCR'''<br>
Oligo bridges initially 30nm pellet.
Diluted down to 30nM in 25μL reaction volume (Added 300μL H<sub>2</sub>O to IDT tube with pelleted DNA to yield 100μM, added 3μL of that dilution to 97μL H<sub>2</sub>O to yield 3μM, added 5μL of that dilution to 45μL H<sub>2</sub>O to yield 300nM, added 2.5μL of this dilution to the 25μL reaction volume for LCR).<br>
<br>
<br>
'''Dephos XbaI linearized MV10 gel purification product'''<br>
 
{| {{table}} border="1" cellspacing="3"
<!-- Editing: the coding for this table is a bit more advanced. -->
<!-- valign="top" aligns all the text in the first row to the top.  -->
<!-- The | symbols on the next two lines start new cells in the same row. This does the same thing as ||, but you have to use | on new lines to set formatting for cells. -->
<!-- bgcolor=#cfcfcf *colors* the *background* of the Reagent and Volume cells grey.  -->
<!-- The next two "rowspan=7" cells span  all 7 rows in the table so that they can fit the "Expected" list and a gel image. rowspan is how you create merged rows in Wiki code. -->
<!-- Replace Plasmid 1 and 2 with the names of your plasmids. Replace insert size and vector size with appropriate information for your plasmids. If you only have one Plasmid, delete all the text for Plasmid 2
<!-- After you upload your gel image to OWW, replace "GelImage.jpg" with the name of your image file -->
<!-- &nbsp; is ASCII code for an invisible space. -->
|- valign="top"
|-
| XbaI gel purified MV10 || 5.0 μL
|-
| 10X Phosphotase buffer || 2.0 μL
|-
| Phosphotase || 1.0 μL
|-
| dH<sub>2</sub>O || 12.0 μL
|-
| Total || 20 μL --> 37°C/ 10 min. 75°C for 2 min.
|}
 
'''LCR Calculations'''<br>
* 1:1 vector to insert
*BL01: (2520bp/5197bp)(1)(50ng)=<u>24.24ng</u>
*<u>24.24ng</u>(1μL/11.3ng)=<u>2.145μL XbaI/SpeI cut BL05 Sample2</u>
*BL05: (2520bp/5197bp)(1)(50ng)=<u>24.24ng</u>
*<u>24.24ng</u>(1μL/18.5ng)=<u>1.31μL XbaI/SpeI cut BL05 Sample1</u>
*50ngCMV/MV9(1μL/59ng)=<u>0.847μL XbaI cut dephos'd CMV/MV9</u>
*50ngCMV/MV9(1μL/15ng)=<u>3.33μL XbaI cut dephos'd CMV/MV9</u>
 
 
 
{| class="wikitable" width=400px align="left"
| &nbsp; || BL05.1|| BL05.2 || Neg
|-
| Insert DNA || 1.3μL || 2.2μL || 0μL
|-
| XbaI Cut Dephos'd CMV/MV9 || 1μL || 3.33μL || 0μL
|-
| Oligo Bridge1 || 2.5μL || 2.5μL || 2.5μL
|-
| Olido Bridge2 || 2.5μL || 2.5μL || 2.5μL
|-
| 10X Ampligase Buffer || 2.5 μl || 2.5μL ||2.5μL
|-
| Ampligase || 1μl || 1μL || 1μL
|-
| Betaine || 0μL || 0μL || 0μL
|-
| DMSO || 0μL || 0μL || 0μL
|-
| dH<sub>2</sub>O || 14.2μL || 11μL || 14.5μL
|-
| Total || 25.0 μL &nbsp;&nbsp;|| 25μL
|-
| colspan="3" | Mix the reaction(s) thoroughly by flicking the tube.<br> Placed in thermocycler on LCR setting. *Volume reduced to ~10μL after removal from PCR machine. Settings incorrect?
|}
<br>
<br>
<br>
<br>
<br>
<br>
<br>
<br>
<br>
<br>
<br>
<br><br>
<br>
<br>
<br>
<br>
<br>
<br>
<br>
'''Long Transformation'''
<br>
Using the set up ligation products from above.
* Warmed selection agar plates at 37°C.
*DH5α-T cells were thawed in ice.
*1.5mL tubes were setup and labeled.
*60μL of thawed cells were mixed and then transferred into each of these two empty tubes.
*The ligation products were then transferred into their respective tubes and flicked to mix. Set back on ice.
*All tubes incubated on ice for 35 minutes.
*All tubes were heat shocked on the heat block at 42°C for 30 seconds and then placed back on ice for 2 minutes.
*900μL SOC Medium was pipetted into each of the three tubes.
*Tubes were taped into an empty plate. The plate was taped to the shaker within the incubator.
*Incubated on shaker for 1 hour at 37°C and 240rpm.
*Centrifuged for 1.5 minutes at 9 x g.
*500μL media removed from each tube.
*Resuspended cell pellet in remaining 400μL media.
*300μL cells transferred onto respective plate.
*Sterile glass beads used to spread cells onto plate.
*Placed in the incubator for overnight growth at 37°C.
<br>




Revision as of 09:37, 26 March 2015



 		<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>

Summary

  • LCR
  • Met Kodibagkar grad student Shubanghi

LCR
Oligo bridges initially 30nm pellet. Diluted down to 30nM in 25μL reaction volume (Added 300μL H2O to IDT tube with pelleted DNA to yield 100μM, added 3μL of that dilution to 97μL H2O to yield 3μM, added 5μL of that dilution to 45μL H2O to yield 300nM, added 2.5μL of this dilution to the 25μL reaction volume for LCR).


Dephos XbaI linearized MV10 gel purification product

XbaI gel purified MV10 5.0 μL
10X Phosphotase buffer 2.0 μL
Phosphotase 1.0 μL
dH2O 12.0 μL
Total 20 μL --> 37°C/ 10 min. 75°C for 2 min.

LCR Calculations

  • 1:1 vector to insert
  • BL01: (2520bp/5197bp)(1)(50ng)=24.24ng
  • 24.24ng(1μL/11.3ng)=2.145μL XbaI/SpeI cut BL05 Sample2
  • BL05: (2520bp/5197bp)(1)(50ng)=24.24ng
  • 24.24ng(1μL/18.5ng)=1.31μL XbaI/SpeI cut BL05 Sample1
  • 50ngCMV/MV9(1μL/59ng)=0.847μL XbaI cut dephos'd CMV/MV9
  • 50ngCMV/MV9(1μL/15ng)=3.33μL XbaI cut dephos'd CMV/MV9


  BL05.1 BL05.2 Neg
Insert DNA 1.3μL 2.2μL 0μL
XbaI Cut Dephos'd CMV/MV9 1μL 3.33μL 0μL
Oligo Bridge1 2.5μL 2.5μL 2.5μL
Olido Bridge2 2.5μL 2.5μL 2.5μL
10X Ampligase Buffer 2.5 μl 2.5μL 2.5μL
Ampligase 1μl 1μL 1μL
Betaine 0μL 0μL 0μL
DMSO 0μL 0μL 0μL
dH2O 14.2μL 11μL 14.5μL
Total 25.0 μL    25μL
Mix the reaction(s) thoroughly by flicking the tube.
Placed in thermocycler on LCR setting. *Volume reduced to ~10μL after removal from PCR machine. Settings incorrect?





















Long Transformation
Using the set up ligation products from above.

  • Warmed selection agar plates at 37°C.
  • DH5α-T cells were thawed in ice.
  • 1.5mL tubes were setup and labeled.
  • 60μL of thawed cells were mixed and then transferred into each of these two empty tubes.
  • The ligation products were then transferred into their respective tubes and flicked to mix. Set back on ice.
  • All tubes incubated on ice for 35 minutes.
  • All tubes were heat shocked on the heat block at 42°C for 30 seconds and then placed back on ice for 2 minutes.
  • 900μL SOC Medium was pipetted into each of the three tubes.
  • Tubes were taped into an empty plate. The plate was taped to the shaker within the incubator.
  • Incubated on shaker for 1 hour at 37°C and 240rpm.
  • Centrifuged for 1.5 minutes at 9 x g.
  • 500μL media removed from each tube.
  • Resuspended cell pellet in remaining 400μL media.
  • 300μL cells transferred onto respective plate.
  • Sterile glass beads used to spread cells onto plate.
  • Placed in the incubator for overnight growth at 37°C.




Retrieved from "https://openwetware.org/mediawiki/index.php?title=Haynes_Lab:Notebook/Engineering_PC-TFs/2015/03/23&oldid=875525"