Haynes Lab:Notebook/Engineering PC-TFs/2013/04/20: Difference between revisions

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(Autocreate 2013/04/20 Entry for Haynes_Lab:Notebook/Engineering_PC-TFs)
 
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==Summary==
==Summary==
*  
=4/2/13==
*1. fshPCD (vector) + BL01
*2. fshPCD (vector) + BL05
*3. flyPCD (vector + BL01
*4. flyPCD (vector) + BL09
 
{|border="1" cellpadding="5" cellspacing="0" align="left"
|-
! scope="col" style="background:#efefef;" | #
! scope="col" style="background:#efefef;" | Biobrick
! scope="col" style="background:#efefef;" | ng/µL
|-
|1
|hPCD : mCh : SP1AB (BL01)
|18.834
|-
|2
|fshPCD : mCh : SP1AB (BL05)
|14.471
|-
|3
|flyPCD : mCh : SP1A (BL09)
|7.302
|-
|4
|hPCD : BL01 (BL12)
|10.84
|-
|}<br><br><br><br><br><br><br><br>
 
{| {{table}}
|align="center" style="background:#f0f0f1;"|'''µL of insert = (length insert/lengthvector *2*25 ng vector/concentration insert)'''
|}
 
{| {{table}}
| align="center" style="background:#0095B6;"|
| align="center" style="background:#0095B6;"|'''1'''
| align="center" style="background:#0095B6;"|'''2'''
| align="center" style="background:#0095B6;"|'''3'''
| align="center" style="background:#0095B6;"|'''4'''
| align="center" style="background:#0095B6;"|'''5'''
|-
| DNA Insert||1.4||1.8||2.0||2.5||--
|-
| DNA Vector (Kozak)||0.3||0.3||0.3||0.3||0.3
|-
| T4 Ligase||1||1||1||1||1
|-
| Lign Buffer (2x)||5||5||5||5||5
|-
| dH2O||2.3||1.9||2.3||1.9||4.7
|-
| ||10 µL||10 µL||10 µL||10 µL||10 µL
|-
|
|}
 
* In a .5 mL tube, pipette the ligation buffer first.
* Then water, DNA insert, vector, and T4 ligase.
* Once completed, allow them to incubate for 10 minutes.
* Meanwhile, get ice bucket and allow competent E. coli cells to thaw for five minutes.
* Then follow transformation process:
'''Transformation Process'''
*Warm five 100 μg/mL Amp agar plates at 37 °C
*Thaw fresh tube of DH5α Turbo cells on ice
*Add 30 μL of DH5α Turbo cells to DNA + dH2O
*Include #5 tube, water only, no DNA (negative control)
*Incubate cells + DNA on ice for 10 min.
*Label pre-warmed plates
*Transfer cells + DNA onto agar
*Add 10 - 15 sterile glass beads, shake, discard beads
*Incubate plates at 37 °C overnight
 
[[Image:plates4_22_2013.jpg|200px|thumb|left|Ligation attempt 4/2/2013]]
*''No colonies on any of the plates. Used cells that are no longer working.''
 




Revision as of 23:53, 22 April 2013

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Summary

4/2/13=

  • 1. fshPCD (vector) + BL01
  • 2. fshPCD (vector) + BL05
  • 3. flyPCD (vector + BL01
  • 4. flyPCD (vector) + BL09
# Biobrick ng/µL
1 hPCD : mCh : SP1AB (BL01) 18.834
2 fshPCD : mCh : SP1AB (BL05) 14.471
3 flyPCD : mCh : SP1A (BL09) 7.302
4 hPCD : BL01 (BL12) 10.84








µL of insert = (length insert/lengthvector *2*25 ng vector/concentration insert)
1 2 3 4 5
DNA Insert 1.4 1.8 2.0 2.5 --
DNA Vector (Kozak) 0.3 0.3 0.3 0.3 0.3
T4 Ligase 1 1 1 1 1
Lign Buffer (2x) 5 5 5 5 5
dH2O 2.3 1.9 2.3 1.9 4.7
10 µL 10 µL 10 µL 10 µL 10 µL
  • In a .5 mL tube, pipette the ligation buffer first.
  • Then water, DNA insert, vector, and T4 ligase.
  • Once completed, allow them to incubate for 10 minutes.
  • Meanwhile, get ice bucket and allow competent E. coli cells to thaw for five minutes.
  • Then follow transformation process:

Transformation Process

  • Warm five 100 μg/mL Amp agar plates at 37 °C
  • Thaw fresh tube of DH5α Turbo cells on ice
  • Add 30 μL of DH5α Turbo cells to DNA + dH2O
  • Include #5 tube, water only, no DNA (negative control)
  • Incubate cells + DNA on ice for 10 min.
  • Label pre-warmed plates
  • Transfer cells + DNA onto agar
  • Add 10 - 15 sterile glass beads, shake, discard beads
  • Incubate plates at 37 °C overnight
Ligation attempt 4/2/2013
  • No colonies on any of the plates. Used cells that are no longer working.



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