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

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(4/20/13)
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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"
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|-
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! scope="col" style="background:#efefef;" | #
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! scope="col" style="background:#efefef;" | Biobrick
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! scope="col" style="background:#efefef;" | ng/µL
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|-
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|1
 +
|hPCD : mCh : SP1AB (BL01)
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|18.834
 +
|-
 +
|2
 +
|fshPCD : mCh : SP1AB (BL05)
 +
|14.471
 +
|-
 +
|3
 +
|flyPCD : mCh : SP1A (BL09)
 +
|7.302
 +
|-
 +
|4
 +
|hPCD : BL01 (BL12)
 +
|10.84
 +
|-
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|}<br><br><br><br><br><br><br><br>
 +
 
 +
{| {{table}}
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|align="center" style="background:#f0f0f1;"|'''µL of insert = (length insert/lengthvector *2*25 ng vector/concentration insert)'''
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|}
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{| {{table}}
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| align="center" style="background:#0095B6;"|
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| align="center" style="background:#0095B6;"|'''1'''
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| align="center" style="background:#0095B6;"|'''2'''
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| align="center" style="background:#0095B6;"|'''3'''
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| align="center" style="background:#0095B6;"|'''4'''
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| align="center" style="background:#0095B6;"|'''5'''
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|-
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| DNA Insert||1.4||1.8||2.0||2.5||--
 +
|-
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| DNA Vector (Kozak)||0.3||0.3||0.3||0.3||0.3
 +
|-
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| T4 Ligase||1||1||1||1||1
 +
|-
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| Lign Buffer (2x)||5||5||5||5||5
 +
|-
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| dH2O||2.3||1.9||2.3||1.9||4.7
 +
|-
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| ||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 01:53, 23 April 2013

Engineering PC-TFs Main project page
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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 Insert1.41.82.02.5--
DNA Vector (Kozak)0.30.30.30.30.3
T4 Ligase11111
Lign Buffer (2x)55555
dH2O2.31.92.31.94.7
10 µL10 µL10 µL10 µL10 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
Ligation attempt 4/2/2013
  • No colonies on any of the plates. Used cells that are no longer working.



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