Turkey/ Protocols

From 2007.igem.org

(Difference between revisions)
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[edit]
'''Growing colonies in broth:'''
'''Growing colonies in broth:'''
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*1. Centrifuge the falcons at 4000rpm for 4-10 minutes.
*1. Centrifuge the falcons at 4000rpm for 4-10 minutes.
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*2. Resuspend pelleted bacterial cells in 250ML Buffer P1(kept in +4C) and transfer to a 1.5mL eppendorf.
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*2. Resuspend pelleted bacterial cells in 250uL Buffer P1(kept in +4C) and transfer to a 1.5mL eppendorf.
-
*3. Add 250ML(microliter) Buffer P2 and invert the tube for ~6 times to mix (do not vortex). Solution should become blue (if indicator is added).
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*3. Add 250uL(microliter) Buffer P2 and invert the tube for ~6 times to mix (do not vortex). Solution should become blue (if indicator is added).
-
*4. Add 350ML Buffer N3 and invert the tube immediately for ~6 times. Solution should become wtite and cloudy.
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*4. Add 350uL Buffer N3 and invert the tube immediately for ~6 times. Solution should become wtite and cloudy.
*5. Centrifuge for 10 minutes at 13000 rpm. A compact white pellet will be formed.
*5. Centrifuge for 10 minutes at 13000 rpm. A compact white pellet will be formed.
*6. Pour the supernatant to Qiagen spin column and centrifuge the column for 1 min. Discard the flow-through.
*6. Pour the supernatant to Qiagen spin column and centrifuge the column for 1 min. Discard the flow-through.
*7. Wash the spin column by adding 0.75mL Buffer PE and centrifuge for 1 min.
*7. Wash the spin column by adding 0.75mL Buffer PE and centrifuge for 1 min.
*8. Discard the flow-through and centrifuge for an additional 6.5-7min to remove residual ethanol in the wash buffer.
*8. Discard the flow-through and centrifuge for an additional 6.5-7min to remove residual ethanol in the wash buffer.
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*9. Place the Qiagen prep column in a clean 1.5 mL eppendorf. Add 32ML (can be modified according to the concentration aimed to be obtained) Buffer EB or water to the C '''center''' of each Qiagen prep spin column and let stand for 5-10 minutes. Centrifuge for 1 min.
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*9. Place the Qiagen prep column in a clean 1.5 mL eppendorf. Add 32uL (can be modified according to the concentration aimed to be obtained) Buffer EB or water to the C '''center''' of each Qiagen prep spin column and let stand for 5-10 minutes. Centrifuge for 1 min.
Store the minipreps at -20. The concentration obtained can be measured by Nanodrop.
Store the minipreps at -20. The concentration obtained can be measured by Nanodrop.
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[edit]
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'''Digestion'''
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'''Digestion:'''
 +
 
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For Vector: Mix (to a total of 20 uL):
 +
7-7.5 uL mini-prepped vector DNA
 +
7.5 uL distilled water
 +
0.3 uL restriction enzyme 1 at 20 units/uL
 +
0.3 uL restriction enzyme 2 at 20 units/uL
 +
0.4 uL calf intestinal alkaline phosphatase (CIP) to prevent re-ligation of the vector to itself
 +
2 uL 10x BSA
 +
2 uL 10x appropriate NEB buffer (check from www.neb.com Double Digest Finder)
 +
 
 +
Insert: Mix (to a total of 10 uL):
 +
7 uL mini-prepped 'vector' DNA
 +
8.2 uL distilled water
 +
0.4 uL restriction enzyme 1 at 20 units/uL
 +
0.4 uL restriction enzyme 2 at 20 units/uL
 +
2 uL 10x BSA
 +
2 uL 10x appropriate NEB buffer
 +
*Incubate overnight at 37C.
 +
*Gel purify the insert using a Qiagen kit. Elute using 20 uL.
 +
*PCR purify the vector (can be gel purified too). Elute in 20 uL.
 +
 
 +
''' Ligation (with Roche Rapid Ligation kit):'''
 +
Mix:
 +
1 uL digested vector
 +
3 uL digested insert
 +
*The optimum molar ratio is 1:3, the volumes can be modified according to concentrations of the vector and insert. For sticky end ligations 1:5 ratio can be used.
 +
*Complete to 10 uL with 1X reagent 2 of the kit (diluted from 5X with distilled water), Vortex and spin.
 +
*Add 10 uL reagent #1.
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*Add 1 uL reagent #3.
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*Ligate for 10 minutes at room temperature.
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*Transform 2 uL of ligation mix in 25 uL DH5alpha competent cells.
 +
 
 +
'''Transformation'''
 +
*Thaw competent E. coli in ice. Take 25uL cells in prechilled eppendorfs. Slowly add 2uL plasmid DNA.
 +
*Incubate in ice for 30 minutes (or more).
 +
*Heat shock at 42C water bath for 30 seconds (timing is critical).
 +
*Incubate for 5 min in ice

Revision as of 00:36, 14 August 2007

[edit] Growing colonies in broth:

  • 1. Prepare 5mL LB + Amp broth in a sterile Falcon tube.
  • 2. Pick up a colony from the plate by a micropipette tip or sterile toothpick and put it in the falcon.
  • 3. Incubate at 37C incubator for 14-16 hours.

Miniprep Plasmid Isolation (with Qiagen kit):

  • 1. Centrifuge the falcons at 4000rpm for 4-10 minutes.
  • 2. Resuspend pelleted bacterial cells in 250uL Buffer P1(kept in +4C) and transfer to a 1.5mL eppendorf.
  • 3. Add 250uL(microliter) Buffer P2 and invert the tube for ~6 times to mix (do not vortex). Solution should become blue (if indicator is added).
  • 4. Add 350uL Buffer N3 and invert the tube immediately for ~6 times. Solution should become wtite and cloudy.
  • 5. Centrifuge for 10 minutes at 13000 rpm. A compact white pellet will be formed.
  • 6. Pour the supernatant to Qiagen spin column and centrifuge the column for 1 min. Discard the flow-through.
  • 7. Wash the spin column by adding 0.75mL Buffer PE and centrifuge for 1 min.
  • 8. Discard the flow-through and centrifuge for an additional 6.5-7min to remove residual ethanol in the wash buffer.
  • 9. Place the Qiagen prep column in a clean 1.5 mL eppendorf. Add 32uL (can be modified according to the concentration aimed to be obtained) Buffer EB or water to the C center of each Qiagen prep spin column and let stand for 5-10 minutes. Centrifuge for 1 min.

Store the minipreps at -20. The concentration obtained can be measured by Nanodrop.

[edit]

Digestion:

For Vector: Mix (to a total of 20 uL): 7-7.5 uL mini-prepped vector DNA 7.5 uL distilled water 0.3 uL restriction enzyme 1 at 20 units/uL 0.3 uL restriction enzyme 2 at 20 units/uL 0.4 uL calf intestinal alkaline phosphatase (CIP) to prevent re-ligation of the vector to itself 2 uL 10x BSA 2 uL 10x appropriate NEB buffer (check from www.neb.com Double Digest Finder)

Insert: Mix (to a total of 10 uL): 7 uL mini-prepped 'vector' DNA 8.2 uL distilled water 0.4 uL restriction enzyme 1 at 20 units/uL 0.4 uL restriction enzyme 2 at 20 units/uL 2 uL 10x BSA 2 uL 10x appropriate NEB buffer

  • Incubate overnight at 37C.
  • Gel purify the insert using a Qiagen kit. Elute using 20 uL.
  • PCR purify the vector (can be gel purified too). Elute in 20 uL.

Ligation (with Roche Rapid Ligation kit): Mix: 1 uL digested vector 3 uL digested insert

  • The optimum molar ratio is 1:3, the volumes can be modified according to concentrations of the vector and insert. For sticky end ligations 1:5 ratio can be used.
  • Complete to 10 uL with 1X reagent 2 of the kit (diluted from 5X with distilled water), Vortex and spin.
  • Add 10 uL reagent #1.
  • Add 1 uL reagent #3.
  • Ligate for 10 minutes at room temperature.
  • Transform 2 uL of ligation mix in 25 uL DH5alpha competent cells.

Transformation

  • Thaw competent E. coli in ice. Take 25uL cells in prechilled eppendorfs. Slowly add 2uL plasmid DNA.
  • Incubate in ice for 30 minutes (or more).
  • Heat shock at 42C water bath for 30 seconds (timing is critical).
  • Incubate for 5 min in ice
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