Team:CU-Boulder/Notebook/Protocols

From 2014.igem.org

(Difference between revisions)
(→Bacterial Conjugation)
(→Isolation of single-stranded phagemid DNA using M13K07 Helper Phage)
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'''Day 1'''
'''Day 1'''
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1.     Add a fresh colony to 50mL LB in 125mL flask. Grow at 37°C, 250rpm until OD is between 0.03 and 0.05
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*1.Add a fresh colony to 50mL LB in 125mL flask. Grow at 37°C, 250rpm until OD is between 0.03 and 0.05
-
 
+
*2.Add M13KO7 helper phage to a final concentration of 1 x10^8 phage/mL*
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2.     Add M13KO7 helper phage to a final concentration of 1 x10^8 phage/mL*
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*3.Continue shaking for 60-90 minutes
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+
*4.Add Kanamycin to final concentration of 70ug/mL
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3.     Continue shaking for 60-90 minutes
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*5.Grow for 14-18 hours at 37°C, 250rpm
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+
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4.     Add Kanamycin to final concentration of 70ug/mL
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-
 
+
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5.     Grow for 14-18 hours at 37°C, 250rpm
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* *Can use a different helper phage if needed. In step 4, add the antibiotic specific to the Helper Phagemid
* *Can use a different helper phage if needed. In step 4, add the antibiotic specific to the Helper Phagemid
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'''Day 2'''
'''Day 2'''
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1.     Spin culture at 4,000 x g for 10 minutes
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*1.Spin culture at 4,000 x g for 10 minutes
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+
*2.Transfer supernatant to a fresh conical. Repeat spin on supernatant
-
2.     Transfer supernatant to a fresh conical. Repeat spin on supernatant
+
*3.Transfer the upper 90% of supernatant to a new conical
-
 
+
*4.Add 0.2 volume of 2.5M NaCl/20% PEG-8000 to the new conical. Gently mix
-
3.     Transfer the upper 90% of supernatant to a new conical
+
*5.Incubate at 4°C for at least 60 minutes
-
 
+
*6.Centrifuge at 12,000 x g for 10 minutes. Carefully decant
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4.     Add 0.2 volume of 2.5M NaCl/20% PEG-8000 to the new conical. Gently mix
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*7.Spin again briefly
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+
*8.Gently resuspend pellet in 1.6mL 1x TBS
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5.     Incubate at 4°C for at least 60 minutes
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*9.Aliquot 800ul into 2 microfuge tubes. Proceed with both tubes
-
 
+
*10.Spin sample for 1 minute to pellet remaining cells. Transfer supernatant to fresh tubes
-
6.     Centrifuge at 12,000 x g for 10 minutes. Carefully decant
+
*11.Add 160ul of 2.5M NaCl/20% PEG-8000 solution to each
-
 
+
*12.Let sit at room temperature for 5 minutes
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7.     Spin again briefly
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*13.Spin at 1300 x g for 10 minutes
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+
*14.Decant the supernatant
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8.     Gently resuspend pellet in 1.6mL 1x TBS
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*15.Spin briefly. Remove supernatant with pipet
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+
*16.Resuspend pellets in 300ul TE
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9.     Aliquot 800ul into 2 microfuge tubes. Proceed with both tubes
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*17.Phenol extraction: add 300ul phenol. Vortex for 15 seconds
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**a.Let sit for 15 minutes. Spin for 10 minutes
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10.   Spin sample for 1 minute to pellet remaining cells. Transfer supernatant to fresh tubes
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*18.Add H2O so volume samples is about 300ul
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+
*19.Phenol/chloroform extraction*: add 300ul PCIA
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11.   Add 160ul of 2.5M NaCl/20% PEG-8000 solution to each
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**a.Vortex for 15 seconds. Spin for 10 minutes
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+
*20.Repeat Phenol/Chloroform extraction
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12.   Let sit at room temperature for 5 minutes
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*21.Chloroform extraction*: add 300ul chloroform
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**a.Vortex 15 seconds. Spin for 10 minutes
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13.   Spin at 1300 x g for 10 minutes
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*22.Add 30ul 2.5M NaAc (pH 4.8)
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+
*23.Add 2-2.5 volumes ethanol
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14.   Decant the supernatant
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*24.Let precipitate for ~2 hours at -20°C
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+
*25.Spin for 1 minute
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15.   Spin briefly. Remove supernatant with pipet
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*26.Decant supernatant
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*27.Resuspend in 25-50ul TE
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16.   Resuspend pellets in 300ul TE
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-
 
+
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17. Phenol extraction: add 300ul phenol. Vortex for 15 seconds
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-
 
+
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*a.       Let sit for 15 minutes. Spin for 10 minutes
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-
 
+
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18. Add H2O so volume samples is about 300ul
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-
 
+
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19. Phenol/chloroform extraction*: add 300ul PCIA
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-
 
+
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*a.       Vortex for 15 seconds. Spin for 10 minutes
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-
 
+
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20. Repeat Phenol/Chloroform extraction
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-
 
+
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21. Chloroform extraction*: add 300ul chloroform
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*a.       Vortex 15 seconds. Spin for 10 minutes
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-
 
+
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22. Add 30ul 2.5M NaAc (pH 4.8)
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+
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23. Add 2-2.5 volumes ethanol
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-
 
+
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24. Let precipitate for ~2 hours at -20°C
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-
 
+
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25. Spin for 1 minute
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-
 
+
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26. Decant supernatant
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-
 
+
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27. Resuspend in 25-50ul TE
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* *Performing steps at 4°C helps with separation
* *Performing steps at 4°C helps with separation
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-
 
'''2.5M NaCl/20% PEG-8000 (5x)'''
'''2.5M NaCl/20% PEG-8000 (5x)'''
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*PEG-800   100g
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*100 g PEG-800
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*NaCl   75g
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*75 g NaCl
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*H<sub>2</sub>O   400mL
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*400 mL H<sub>2</sub>O
** *Bring final volume to 500 mL  
** *Bring final volume to 500 mL  

Revision as of 04:37, 4 October 2014


Contents

Amplification of Phage using Helper Phage

Need

  • Plate of infectable cells that contain F’ episome
  • 2.5M NaCl/20% PEG-8000
  • 1x TBS

Day 1

  • 1.Add a fresh colony of infectable cells to 50mL LB in 125mL flask.
    • a. Include phagemid antibiotic only.
    • b. Grow at 37°C, 250rpm until OD is between 0.03 and 0.05
  • 2.Add the helper phage to a final concentration of 1 x10^8 phage/mL
  • 3.Incubate for 60-90 minutes, shaking
  • 4.Add Helper Phagemid antibiotic to a high concentration
  • 5.Grow for 14-18 hours at 37°C, shaking

Day 2

  1. Spin culture at 4,000 x g for 10 minutes
  2. Transfer supernatant to a fresh conical. Repeat spin on supernatant
  3. Transfer the upper 90% of supernatant to a new conical
  4. Add 0.2 volume of 2.5M NaCl/20% PEG-8000 to the new conical. Gently mix
  5. Incubate at 4°C for at least 60 minutes
  6. Centrifuge at 12,000 x g for 10 minutes. Carefully decant
  7. Spin again briefly
  8. Gently resuspend pellet in 1.6mL 1x TBS
  9. Aliquot 800ul into 2 microfuge tubes. Proceed with both tubes
  10. Spin sample for 1 minute to pellet remaining cells. Transfer supernatant to fresh tubes
  11. Add 160ul of 2.5M NaCl/20% PEG-8000 solution to each
  12. Let sit at room temperature for 5 minutes
  13. Spin at 1300 x g for 10 minutes
  14. Decant the supernatant
  15. Spin briefly. Remove supernatant with pipet
  16. Resuspend pellet in 200ul 1x TBS.
    • a. If desired, combine contents of both tubes into one

2.5M NaCl/20% PEG-8000 (5x)

  • 100 g PEG-8000
  • 75 g NaCl
  • 400 mL H2O
  • Bring final volume to 500 mL

TBS (1x)

  • 6.05 g Tris
  • 8.76 g NaCl
  • 800 mL H2O
  • Adjust pH to 7.6 with 1M HCL
  • Adjust volume to 1 L




Bacterial Transformation Using Frozen Competent Cells

Before you start

  • Heat hot plate or water bath to 42°C
  • Warm selection plates to 37°C

Transformation

  • 1.Thaw chemically competent cells on ice for 10-15 minutes
  • 2.Add 40ul cells to fresh 1.7mL tube
  • 3.Add DNA
    • a. If using a ligation product add up to 10ul of sample
    • b. If using supercoiled plasmid add 100ng
  • 4.Incubate on ice for 30 minutes
  • 5.Heat shock cells on hot plate (or water bath) for 30-45s* @ 42°C
  • 6.Incubate on ice for 2-5 minutes
  • 7.Add 200 μL SOC and shake gently for 1-2 hours @ 37° C
    • a.(Note: Can also recover in 960ul. After recovery, gently spin cells and remove supernatant. Resuspend in 200ul LB)
  • 8.Plate 100-200ul cells onto selection plates
    • a. If high efficiency is expected, we suggest also plating a 1:10 dilution
  • 9.Once dry, turn upside down (agar on top) and incubate overnight @ 37° C
    • *Optimal timing depends on cells


SOC (1L)

  • 20 g Tryptone
  • 5 g YeastExtract
  • 4.8 g MgSO4
  • 3.6 g Dextrose
  • 0.5 g NaCl
  • 0.186 g KCL




Bacterial Conjugation

Need

  • Donor cells: Cells already containing F’ episome
  • Recipient cells: Cells with a resistance marker that is absent from donor cells
  • Double selection plate containing antibiotic to select for F’ episome and a second antibiotic to select for the recipient cells

Day 1

  1. Set up liquid overnight of donor cells. Include antibiotic
  2. Set up liquid overnight of recipient cells. Include antibiotic

Day 2

  • 1.Mix 500ul of each overnight sample in a new tube. Mix by pipetting or flicking
  • 2.Incubate for 30 minutes at 37°C, shaking
  • 3.Plate 100ul onto double selection plate
    • a.We advise also plating a 1:10 dilution
  • 4.Incubate at 37°C

http://inst.bact.wisc.edu/inst/index.php?module=Book&func=displayarticle&art_id=128


Making Chemically Competent Bacteria

To prepare for Day 2

  • Set centrifuge to 4°C
  • MgCl2 and CaCl2 solutions
  • Thaw DMSO
  • Chill tubes
  • Acquire liquid nitrogen or dry ice

Day 1

  1. Grow cells O/N

Day 2

  • 1.Add 0.5mL of the overnight culture to 50mL LB
  • 2.Grow until OD is between 0.2 and 0.4
  • 3.Incubate on ice for 30 minutes
  • 4.Centrifuge for 10 minutes at 2700 x g and 4°C
  • 5.Decant. Dry upside down on a paper towel for 1 minute
  • 6.Completely resuspend in 30mL 0.8M MgCl2, 0.2M CaCl2
    • a. Gently vortex
  • 7.Centrifuge for 10 minutes at 2700 x g and 4°C
  • 8.Decant. Dry upside down on a paper towel for 1 minute
  • 9.Fully resuspend in 2mL of 0.1M CaCl2
  • 10.Chill sample on ice. Add 70ul DMSO, keeping the sample tube on ice
  • 11.Swirl to mix
  • 12.Incubate on ice for 15 minutes
  • 13.Add 70ul DMSO, swirl to mix, keeping the sample tube on ice
  • 14.Dispense 200ul into pre-chilled 1.7mL tubes
  • 15.Snap freeze with liquid nitrogen or dry ice
  • 16.Store at -80°C until ready to use




Isolation of single-stranded phagemid DNA using M13K07 Helper Phage

Need

  • Fresh plate of infectable cells (contain F’ episome)
  • 2.5M NaCl/20% PEG-8000
  • TBS, TE, phenol, phenol/chloroform, chloroform

Day 1

  • 1.Add a fresh colony to 50mL LB in 125mL flask. Grow at 37°C, 250rpm until OD is between 0.03 and 0.05
  • 2.Add M13KO7 helper phage to a final concentration of 1 x10^8 phage/mL*
  • 3.Continue shaking for 60-90 minutes
  • 4.Add Kanamycin to final concentration of 70ug/mL
  • 5.Grow for 14-18 hours at 37°C, 250rpm
  • *Can use a different helper phage if needed. In step 4, add the antibiotic specific to the Helper Phagemid

Day 2

  • 1.Spin culture at 4,000 x g for 10 minutes
  • 2.Transfer supernatant to a fresh conical. Repeat spin on supernatant
  • 3.Transfer the upper 90% of supernatant to a new conical
  • 4.Add 0.2 volume of 2.5M NaCl/20% PEG-8000 to the new conical. Gently mix
  • 5.Incubate at 4°C for at least 60 minutes
  • 6.Centrifuge at 12,000 x g for 10 minutes. Carefully decant
  • 7.Spin again briefly
  • 8.Gently resuspend pellet in 1.6mL 1x TBS
  • 9.Aliquot 800ul into 2 microfuge tubes. Proceed with both tubes
  • 10.Spin sample for 1 minute to pellet remaining cells. Transfer supernatant to fresh tubes
  • 11.Add 160ul of 2.5M NaCl/20% PEG-8000 solution to each
  • 12.Let sit at room temperature for 5 minutes
  • 13.Spin at 1300 x g for 10 minutes
  • 14.Decant the supernatant
  • 15.Spin briefly. Remove supernatant with pipet
  • 16.Resuspend pellets in 300ul TE
  • 17.Phenol extraction: add 300ul phenol. Vortex for 15 seconds
    • a.Let sit for 15 minutes. Spin for 10 minutes
  • 18.Add H2O so volume samples is about 300ul
  • 19.Phenol/chloroform extraction*: add 300ul PCIA
    • a.Vortex for 15 seconds. Spin for 10 minutes
  • 20.Repeat Phenol/Chloroform extraction
  • 21.Chloroform extraction*: add 300ul chloroform
    • a.Vortex 15 seconds. Spin for 10 minutes
  • 22.Add 30ul 2.5M NaAc (pH 4.8)
  • 23.Add 2-2.5 volumes ethanol
  • 24.Let precipitate for ~2 hours at -20°C
  • 25.Spin for 1 minute
  • 26.Decant supernatant
  • 27.Resuspend in 25-50ul TE
  • *Performing steps at 4°C helps with separation


2.5M NaCl/20% PEG-8000 (5x)

  • 100 g PEG-800
  • 75 g NaCl
  • 400 mL H2O
    • *Bring final volume to 500 mL

TBS (1x)

  • 6.05 g Tris
  • 8.76 g NaCl
  • 800 mL H2O
    • *Adjust pH to 7.6 with 1M HCL
    • *Adjust volume to 1L




M13 Amplification


This protocol is to make more M13 phages.

Need

  • Fresh plate of infectable cells (contain F’ episome)

Day 1

  • 1.Grow liquid overnight culture of infectable cells

Day 2

  • 1.Add 200ul overnight culture to 20mL LB in a 250mL flask
  • 2.Add 1ul phage suspension
  • 3.Incubate for 4-5 hours at 37°C, 250rpm
  • 4.Centrifuge for 10 minutes at 4500 x g
  • 5.Transfer supernatant to a new tube.
  • 6.Repeat centrifugation on supernatant
  • 7.Transfer top 16mL of supernatant to a new tube
  • 8.Add 4mL of 2.5M NaCl/20% PEG-8000. Briefly mix
  • 9.Precipitate phage for 1 hour or overnight at 4°C
  • 10.Centrifuge for 15 minutes at 12000 x g. Decant supernatant
  • a. Spin briefly. Remove residual supernatant with pipet
  • 11.Resuspend pellet in 1mL 1x TBS. Transfer to 1.7mL tube
  • 12.Spin briefly to remove cell debris
  • 13.Transfer supernatant to a new tube
  • 14.Add 200ul of 2.5M NaCl/20% PEG-8000
  • 15.Incubate on ice for 15-60 minutes
  • 16.Spin for 10 minutes at 12000-14000 rpm. Discard supernatant
  • 17.Briefly spin. Remove supernatant with pipette
  • 18.Resuspend pellet in 200uL TBS
  • 19.For long term storage at -20C, add 200uL glycerol


2.5M NaCl/20% PEG-8000 (5x)

  • 100 g PEG-8000
  • 75 g NaCl
  • 400 mL H2O
    • Bring final volume to 500 mL


TBS (1x)

  • 6.05 g Tris
  • 8.76 g NaCl
  • 800 mL H2O
Adjust pH to 7.6 with 1M HCl
Adjust volume to 1 L
Store at 3C for up to 3 months




QIAprep Spin Miniprep (Centrifuge method)

  • This protocol is taken from the Qiagen Mini-prep Kit and is used to isolate plasmid DNA from a bacterial overnight.

Notes before starting

  • *Optional: Add LyseBlue reagent to Buffer P1 at a ratio of 1 to 1000
  • *Add the provided RNase A solution to Buffer P1, mix, store bottle at 2-8°C
  • *Add ethanol (96-100%) to Buffer PE before use
  • *All centrifugation steps are carried out at 13,000 rpm (~17,900xg) in a conventional table-top microcentrifuge


  • 1.Centrifuge 1-6mL bacterial overnight culture at >8000 rpm (6800xg) for 3 minutes at room temperature (15-25C)
  • 2.Resuspend pellet in 250ul Buffer P1 and transfer to microcentrifuge tube
  • 3.Add 250ul Buffer P2 and mix thoroughly by inverting the tube 4-6 times until the solution becomes clear.
  • a.DO NOT allow lysis reaction to proceed for more than 5 minutes
  • b.If using LyseBlue reagent, the solution will turn blue
  • 4.Add 350ul Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
  • a.If using LyseBlue reagent, the solution will turn colorless
  • 5.Centrifuge for 10 minutes
  • 6.Apply supernatant from step 5 to the QIAprep spin column by decanting or pipetting. Centrifuge for 30-60 s and discard the flow-through
  • 7.Recommended: Wash the QIAprep spin column by adding 500 ul Buffer PB. Centrifuge for 30-60 s and discard the flow-through
  • a.Only required when using endA+ strains or other bacterial strains with high nuclease activity or carbohydrate content
  • 8.Wash the QIAprep spin column by adding 750ul of Buffer PE. Centrifuge for 30-60 s and discard the flow-through
  • 9.Centrifuge for 1 minutes to remove residual wash buffer
  • 10.Place the QIAprep column in a clean 1.5mL microcentrifuge tube. To elute DNA, add 30ul Buffer EB. Let stand for 1 min, and centrifuge for 1 minute
  • a.Can elute DNA in 50ul but this will decrease DNA concentration
  • b.To increase yield, let sit for up to 4 minutes

Buffer Recipes

P1 50 mM Tris-Cl, pH 8.0 10 mM EDTA 100 ug/mL RNase A

*After RNase A addition, the buffer should be stored at 2-8C

P2 (Lysis buffer)

  • 200 mM NaOH
  • 1% SDS (w/v)

N3*

  • 4.2 M Gu-HCl
  • 0.9 M KAc, pH 4.8

PB*

  • 5 M Gu-HCl
  • 30% isopropanol

PE*

  • 10 mM Tris-HCl pH 7.5
  • 80% ethanol

Elution Buffer (EB)

  • 10 mM Tris-CL, pH 8.5
  • Recipes from OpenWetWare



Retrieved from "http://2014.igem.org/Team:CU-Boulder/Notebook/Protocols"