User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2012/04/06

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(Autocreate 2012/04/06 Entry for User:Behzad_Damadzadeh/Notebook/PcTF_Subcloning_in_E-coli)
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=Date=
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=06/04/12=
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'''Assemblies'''
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# BD112 (final assembly 1): BD111/(X/P)/1138 + BBa_I712074/(S/P)46
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# BD113 (final assembly 2): BD111/(X/P)/1138 + BBa_I719005/(S/P)23
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'''List title'''
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[[Image:T7-RBS-PcTF-Assembly.jpg|thumb|350px|BD112 and BD113 assembly]]
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# List items
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> Digests (Fermentas FD)<br>
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{| class="wikitable" width=400px
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| Plasmid DNA || 20.0 μl*
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|-
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| Fermentas FastDigest enzyme 1 || 1.0 μl
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|-
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| Fermentas FastDigest enzyme 2 || 1.0 μl
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|-
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| 10x FastDigest buffer + green loading dye || 3.0 μl
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|-
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| dH<sub>2</sub>O || 5.0 μl
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|-
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| &nbsp; || 30.0 μl total
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|-
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| colspan="2" | *For low yield DNA, use up to 25 μL; decrease dH<sub>2</sub>O accordingly.<br>Mix the reaction(s) thoroughly by flicking the tube.<br>Incubate at 37°C for 30 minutes.
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|}
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'''Separate the fragments via gel electrophoresis and purify the fragments'''
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[[Image:PcTF-RBS-Digestion-Gel-Behzad4-4-12.jpg|thumb|350px|White dashed lines border where the gel was cut to excise vector fragment (RBS) and insert fragment (PcTF).]]
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# Make a 0.8% gel: add 0.5 g agarose to ~60 ml 1x TAE buffer in a glass flask.
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# Mix by swirling and microwave for 40 seconds. Mix by swirling again (to eliminate air pockets and prevent boiling-over) and microwave for 40 seconds.
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# Set up a gel mold and comb. Make sure the teeth are the right size to hold 30 μL of sample.
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# Add 6 μl of 10 mg/ml ethidium bromide (etBr) to the agarose for a final concentration of ~0.8 μg/mL etBr. Mix by swirling (avoid making bubbles).
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# Pour the gel into the gel mold. Allow it to cool until it becomes opaque.
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# Fill a gel electrophoresis chamber with 1x TAE.
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# Remove the comb from the gel and carefully submerge the gel into the filled electrophoresis chamber.
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# Carefully pipette 15 μL pre-made 1 kb ladder mix into the first empty well and the DNA samples into the other empty wells.
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# Connect the electrical leads so that the positive end is at the bottom (DNA migrates to the positive end). Run the gel at 100 V.
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# Stop the gel when the yellow dye (Orange G) reaches the desired place on the gel (~1 hr.).
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# Remove the gel from the chamber and photograph under UV light.
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# Use a scalpel to cut the appropriate sized band(s) from the gel, place each gel slice in a 1.5 mL tube, and purify the DNA with Zymoclean Gel DNA Recovery Kit(Follow the protocol inside the kit box).
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# Measure the concentration of the purified fragment samples with a Biotek Take3 plate from the Stabenfeldt lab. Record the absorbance (A260), purity (A260/A280), and concentration (ng/μl) for each sample.
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Biotek Take3 Result:
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  PcTF: 15.512 (ng/μl), 2.123 (A260/A280)
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  RBS:  3.201  (ng/μl), 2.462 (A260/A280)

Revision as of 15:37, 7 April 2012

PcTF Subcloning in E. coli Main project page
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06/04/12

Assemblies

  1. BD112 (final assembly 1): BD111/(X/P)/1138 + BBa_I712074/(S/P)46
  2. BD113 (final assembly 2): BD111/(X/P)/1138 + BBa_I719005/(S/P)23
BD112 and BD113 assembly
BD112 and BD113 assembly

> Digests (Fermentas FD)


Plasmid DNA 20.0 μl*
Fermentas FastDigest enzyme 1 1.0 μl
Fermentas FastDigest enzyme 2 1.0 μl
10x FastDigest buffer + green loading dye 3.0 μl
dH2O 5.0 μl
  30.0 μl total
*For low yield DNA, use up to 25 μL; decrease dH2O accordingly.
Mix the reaction(s) thoroughly by flicking the tube.
Incubate at 37°C for 30 minutes.

Separate the fragments via gel electrophoresis and purify the fragments

White dashed lines border where the gel was cut to excise vector fragment (RBS) and insert fragment (PcTF).
White dashed lines border where the gel was cut to excise vector fragment (RBS) and insert fragment (PcTF).
  1. Make a 0.8% gel: add 0.5 g agarose to ~60 ml 1x TAE buffer in a glass flask.
  2. Mix by swirling and microwave for 40 seconds. Mix by swirling again (to eliminate air pockets and prevent boiling-over) and microwave for 40 seconds.
  3. Set up a gel mold and comb. Make sure the teeth are the right size to hold 30 μL of sample.
  4. Add 6 μl of 10 mg/ml ethidium bromide (etBr) to the agarose for a final concentration of ~0.8 μg/mL etBr. Mix by swirling (avoid making bubbles).
  5. Pour the gel into the gel mold. Allow it to cool until it becomes opaque.
  6. Fill a gel electrophoresis chamber with 1x TAE.
  7. Remove the comb from the gel and carefully submerge the gel into the filled electrophoresis chamber.
  8. Carefully pipette 15 μL pre-made 1 kb ladder mix into the first empty well and the DNA samples into the other empty wells.
  9. Connect the electrical leads so that the positive end is at the bottom (DNA migrates to the positive end). Run the gel at 100 V.
  10. Stop the gel when the yellow dye (Orange G) reaches the desired place on the gel (~1 hr.).
  11. Remove the gel from the chamber and photograph under UV light.
  12. Use a scalpel to cut the appropriate sized band(s) from the gel, place each gel slice in a 1.5 mL tube, and purify the DNA with Zymoclean Gel DNA Recovery Kit(Follow the protocol inside the kit box).
  13. Measure the concentration of the purified fragment samples with a Biotek Take3 plate from the Stabenfeldt lab. Record the absorbance (A260), purity (A260/A280), and concentration (ng/μl) for each sample.

Biotek Take3 Result:

 PcTF: 15.512 (ng/μl), 2.123 (A260/A280)
 RBS:  3.201  (ng/μl), 2.462 (A260/A280)
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