Griffitts:Bacterial DNA preparation: Difference between revisions
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==Materials== | ==Materials== | ||
<!-- These are buffers used in previous versions of the protocol: | |||
* [[Griffitts:Bacterial DNA preparation#Buffer B1 (40 mL)|Buffer B1]] | * [[Griffitts:Bacterial DNA preparation#Buffer B1 (40 mL)|Buffer B1]] | ||
* [[Griffitts:Bacterial DNA preparation#Buffer B2 (40 mL)|Buffer B2]] | * [[Griffitts:Bacterial DNA preparation#Buffer B2 (40 mL)|Buffer B2]] | ||
* Qiagen [[Griffitts:Bacterial DNA preparation#Protease|Protease]] (in QIAGEN kit) | |||
* [[Griffitts:Bacterial DNA preparation#Lysozyme (5 mL)|Lysozyme]]--> | |||
* Buffer QBT (in QIAGEN kit) | * Buffer QBT (in QIAGEN kit) | ||
* Buffer QC (in QIAGEN kit) | * Buffer QC (in QIAGEN kit) | ||
* Buffer QF (in QIAGEN kit) | * Buffer QF (in QIAGEN kit), warmed to 50°C | ||
* | * Proteinase K | ||
* [[Griffitts:Bacterial DNA preparation#RNase A (1 mL)|RNase A]] | * [[Griffitts:Bacterial DNA preparation#RNase A (1 mL)|RNase A]] | ||
* Isopropanol (room temperature) | * Isopropanol (at room temperature) | ||
* 70% ethanol ( | * Chloroform (at room temperature) | ||
* [[Griffitts:Common buffers#TE buffer| | * 70% ethanol (at 4°C) | ||
* [[Griffitts:Common buffers#TE buffer|T<small>10</small>E<small>1</small>]] | |||
* [[Griffitts:Bacterial DNA preparation#5 M NaCl (75 mL)|5 M NaCl]] | |||
* [[Griffitts:Bacterial DNA preparation#Binding Buffer|Binding Buffer]] | |||
* [[Griffitts:Bacterial DNA preparation#10% CTAB (10 mL)|10% CTAB]] | |||
* QiaPrep Genomic-tips (1 tip per sample) | * QiaPrep Genomic-tips (1 tip per sample) | ||
* 1.7-mL microcentrifuge | * 1.7-mL microcentrifuge tubes | ||
* 15-mL Falcon tubes ( | * 15-mL Falcon tubes | ||
* [[Griffitts:Stock solutions#10% SDS (75 mL)|10% SDS]] | |||
==Buffer preparation== | ==Buffer preparation== | ||
<!-- The following buffers were used in older versions of the procedure | |||
===Buffer B1 (40 mL)=== | ===Buffer B1 (40 mL)=== | ||
* 32 mL dH<sub>2</sub>O | * 32 mL dH<sub>2</sub>O | ||
Line 36: | Line 44: | ||
* Dissolve lyophilized Qiagen protease in 1.4 mL ddH<sub>2</sub>O | * Dissolve lyophilized Qiagen protease in 1.4 mL ddH<sub>2</sub>O | ||
* Store at 4°C | * Store at 4°C | ||
===Lysozyme (1 mL)=== | ===100 mg/mL Lysozyme (1 mL)=== | ||
* 100 mg lysozyme (in –20°C freezer) | * 100 mg lysozyme (in –20°C freezer) | ||
* 1 mL ddH<sub>2</sub>O | * 1 mL ddH<sub>2</sub>O | ||
* Vortex | * Vortex | ||
* Divide into 100 μL aliquots | * Divide into 100 μL aliquots | ||
* Store at –20°C | * Store at –20°C --> | ||
===RNase A (200 μL)=== | ===50 mg/mL RNase A (200 μL)=== | ||
* 10 mg RNase A (in –20°C freezer) | * 10 mg RNase A (in –20°C freezer) | ||
* 200 μL ddH<sub>2</sub>O | * 200 μL ddH<sub>2</sub>O | ||
Line 48: | Line 56: | ||
* Divide into 20 μL aliquots | * Divide into 20 μL aliquots | ||
* Store at –20°C | * Store at –20°C | ||
===5 M NaCl (75 mL)=== | |||
* 50 mL dH<sub>2</sub>O | |||
* 21.9 g NaCl | |||
* This may require some heating | |||
* QS to volume with dH<sub>2</sub>O | |||
* Autoclave | |||
===10% CTAB (10 mL)=== | |||
* 6 mL ddH<sub>2</sub>O | |||
* 1.4 mL [[Griffitts:Bacterial DNA preparation#5 M NaCl (75 mL)|5 M NaCl]] | |||
* 1 g hexadecyltrimethylammonium bromide (CTAB) | |||
* Alternate vortexing and 10-minute incubations in the 60°C water bath until dissolved | |||
* QS to volume with ddH<sub>2</sub>O | |||
===<!-- Dr. Griffitt's New and Improved -->Binding Buffer<!-- Liquid Liniment -->=== | |||
(This is the Qiagen Equilibration Buffer without isopropanol) | |||
* 1.4 mL [[Griffitts:Stock solutions#5 M NaCl (75 mL)|5 M NaCl]] | |||
* 1 mL [[Griffitts:Common buffers#0.5 M MOPS (40 mL)|0.5 M MOPS]] | |||
* 150 μL [[Griffitts:Common buffers#10% Triton X-100 (40 mL)|10% Triton X-100]] | |||
* 7.45 mL ddH<sub>2</sub>O | |||
==Procedure== | ==Procedure== | ||
<font color=red>'''NOTE: Do NOT use pipetting for resuspension—it can cause shearing of the genomic DNA.'''</font> | <font color=red>'''NOTE: Do NOT use pipetting for resuspension—it can cause shearing of the genomic DNA.'''</font> | ||
===Lysis=== | ===Lysis=== | ||
# [[Griffitts:Bacterial DNA preparation#Buffer preparation|Prepare all buffers and stock solutions beforehand]] and label all tubes | |||
#* All buffers (except Buffer QF and the 70% ethanol) should be equilibrated to room temperature | |||
#* Pre-warm the Buffer QF to 50°C to increase yields | |||
#* 70% ethanol should be cold (4°C) | |||
# Grow ''S. meliloti'' cultures in 4-mL LB overnight to saturation | |||
# Centrifuge two 1.5-mL aliquots of each culture at 13,200 rpm for 2 min. in 1.7-mL Eppendorf tubes | |||
# Discard supernatant | |||
# Thoroughly resuspend each pellet in 1 mL LB | |||
# Centrifuge at 13,200 rpm for 2 minutes | |||
* Add | # Discard supernatant | ||
# Resuspend each pellet in 735 μL [[Griffitts:Common buffers#TE buffer|T<small>10</small>E<small>1</small>]] | |||
* Incubate | # Add 39 μL 10% [[Griffitts:Stock solutions#20% SDS (75 mL)|SDS]] to each tube | ||
* Add | # Invert several times | ||
# Immediately add 5 μL [[Griffitts:Common buffers#Proteinase K|Proteinase K]] to each tube | |||
# Vortex 5 seconds | |||
# Incubate on the rotator at 37°C for 60 minutes <!-- until sample becomes clear? --> | |||
* Remove | * '''Optional:''' Remove 300 μL and save for an analytical gel ('''test aliquot 1''' = nuclear lysate) | ||
===CTAB Genomic DNA Isolation=== | |||
# Add 125 μL [[Griffitts:Bacterial DNA preparation#5 M NaCl (75 mL)|5 M NaCl]] to each tube | |||
# Vortex 10 seconds | |||
# Add 100 μL [[Griffitts:Bacterial DNA preparation#10% CTAB (10 mL)|10% CTAB]] to each tube | |||
#* Make sure the CTAB hasn't crashed out of solution; if it has, alternate vortexing and 10-minute incubations in the 60°C water bath until it is dissolved | |||
#* At this point a precipitate should form | |||
# Vortex 10 seconds | |||
# Incubate on the 60°C for 15 minutes | |||
# Add 500 μL chloroform | |||
# Vortex 10 seconds and shake to make a milky emulsion | |||
# Centrifuge for 20 minutes at maximum speed | |||
#* A well-packed interface should form | |||
# Using a P200, remove ~800 μL of the aqueous layer (top layer) to a new tube | |||
#* Do this slowly so that you don't suck up the debris at the interface | |||
# Add 2 μL [[Griffitts:Bacterial DNA preparation#50 mg/mL RNase A (200 μL)|RNase A]] | |||
# Vortex 5 seconds | |||
# Incubate at 37°C for 20 minutes | |||
* '''Optional:''' Remove 8 μL and save for an analytical gel ('''test aliquot 2''' = CTAB purification) | |||
===Genomic | ===Qiagen Genomic Column=== | ||
# Suspend a QIAGEN Genomic-tip 20/G over a culture tube using a yellow tip holder | |||
# Equilibrate the tip with 2 mL Buffer QBT and allow it to empty by gravity flow | |||
# Add 200 μL [[Griffitts:Bacterial DNA preparation#Binding Buffer|Binding Buffer]] to each 1.7-mL tube | |||
* Add each sample to a tip and allow it to enter the resin by gravity flow | # Combine samples from the same original culture | ||
#* Be careful not to mix samples from different cultures! | |||
* Remove 300 μL and save for an analytical gel ('''aliquot | # Add each sample to a tip and allow it to enter the resin by gravity flow | ||
#* You may have to apply positive pressure by mouth (1 drop every 5–10 seconds) | |||
* Wash | #* '''Optional:''' Remove 300 μL and save for an analytical gel ('''test aliquot 3''' = flow-through fraction) | ||
* Remove | # Move the tip and tip holder to a new culture tube | ||
# Wash with 1 mL of Buffer QC and allow it to move through by gravity flow | |||
#* You may have to apply positive pressure by mouth (1 drop every 5–10 seconds) | |||
* | # Wash again with 2 mL of Buffer QC and allow it to move through by gravity flow | ||
#* You may have to apply positive pressure by mouth (1 drop every 5–10 seconds) | |||
* Centrifuge at maximum | #* '''Optional:''' Remove 1.2 mL and save for an analytical gel ('''test aliquot 4''' = wash fraction) | ||
# Move the tip and tip holder to a new culture tube | |||
# Elute with 2 mL of 50°C Buffer QF and allow it to move through by gravity flow | |||
#* You may have to apply positive pressure by mouth (1 drop every 5–10 seconds) | |||
# Separate the eluate into 2 1.5-mL tubes (1-mL per tube) | |||
# Precipitate the DNA with 700 μL of isopropanol | |||
* | #* At this point and on, you should include your test aliquots if you chose to collect them | ||
** | # Rock gently for 1 minute | ||
# Place tubes on ice for 15 minutes | |||
#* The DNA precipitate should sink to the bottom of the tube | |||
# Centrifuge at maximum speed for 5 minutes <!-- at 4°C? --> | |||
# Remove most of the isopropanol | |||
# Centrifuge at maximum speed for 1 minute <!-- at 4°C? --> | |||
# Remove the rest of the isopropanol with a P-200 | |||
# Add 100 μL cold 70% ethanol | |||
#* Do '''not''' pipette up and down or vortex! | |||
# Centrifuge at maximum speed for 2 minutes <!-- at 4°C? --> | |||
# Remove all of the ethanol with a P-200 | |||
# Add 60 μL of [[Griffitts:Common buffers#TE buffer|T<small>10</small>E<small>1</small>]] | |||
#* Drizzle the [[Griffitts:Common buffers#TE buffer|TE buffer]] twice down the back of the tube | |||
#* Do '''not''' pipette up and down or vortex! | |||
# To dissolve the DNA, incubate at 60°C and flick once every minute for 15 minutes | |||
# Combine DNA samples from the same original culture into one tube | |||
#* Final volume should be 120 μL | |||
===Analysis=== | ===Analysis=== | ||
# Analyze by [[Griffitts:Gel electrophoresis|gel electrophoresis]] on a 1% gel for 30 minutes at 120 V | |||
#* For test aliquots, add 2 μL [[Griffitts:Common buffers#8X DNA loading|8X DNA loading dye]] to 2 μL DNA and load 4 μL | |||
* | #* For final DNA samples, perform a dilution series using 6 μL [[Griffitts:Common buffers#8X DNA loading|8X DNA loading dye]] to 3 μL DNA (or 3 μL of the previous dilution) and load 4 μL | ||
* | #* The final concentration should be at least 5 ng <!-- is that right? --> in no more than 100 μL | ||
* | <!-- # Analyze by Nanodrop | ||
* | #* Concentration should be >200 μL | ||
#* A<small>260</small>/A<small>280</small> should be from 1.7 to 1.9 (evaluates presence of contaminating protein) | |||
#* A<small>260</small>/A<small>230</small> should be >2 (evaluates the presence of contaminating RNA)--> | |||
<br> | <br> | ||
<br> | <br> | ||
<br> | <br> | ||
''Adapted from QIAGEN Genomic DNA handbook.'' | ''Adapted from the QIAGEN Genomic DNA handbook and various CTAB procedures: | ||
:: [http://www.docstoc.com/docs/640147/DNA-Isolation-Bacterial-CTAB-Protocol---General-Info DOE JGI] | |||
:: [http://www.bio.vu.nl/geomicrob/protocols/DNA_isolation/DNA-isol-Bacteria.pdf Current protocols in Molecular Biology (.pdf)] | |||
:: [http://www.springerlink.com/content/g0516q4h678120wu/ The Nucleic Acid Protocols Handbook] | |||
:: [http://www.springerlink.com/content/t7284786x7n153l3/ Protocols for Nucleic Acid Analysis by Nonradioactive Probes] | |||
:: [http://cmgm.stanford.edu/biology/long/protocols.htm#CTAB Sharon Long lab protocols].'' |
Latest revision as of 11:11, 20 May 2010
Materials
- Buffer QBT (in QIAGEN kit)
- Buffer QC (in QIAGEN kit)
- Buffer QF (in QIAGEN kit), warmed to 50°C
- Proteinase K
- RNase A
- Isopropanol (at room temperature)
- Chloroform (at room temperature)
- 70% ethanol (at 4°C)
- T10E1
- 5 M NaCl
- Binding Buffer
- 10% CTAB
- QiaPrep Genomic-tips (1 tip per sample)
- 1.7-mL microcentrifuge tubes
- 15-mL Falcon tubes
- 10% SDS
Buffer preparation
50 mg/mL RNase A (200 μL)
- 10 mg RNase A (in –20°C freezer)
- 200 μL ddH2O
- Vortex
- Divide into 20 μL aliquots
- Store at –20°C
5 M NaCl (75 mL)
- 50 mL dH2O
- 21.9 g NaCl
- This may require some heating
- QS to volume with dH2O
- Autoclave
10% CTAB (10 mL)
- 6 mL ddH2O
- 1.4 mL 5 M NaCl
- 1 g hexadecyltrimethylammonium bromide (CTAB)
- Alternate vortexing and 10-minute incubations in the 60°C water bath until dissolved
- QS to volume with ddH2O
Binding Buffer
(This is the Qiagen Equilibration Buffer without isopropanol)
- 1.4 mL 5 M NaCl
- 1 mL 0.5 M MOPS
- 150 μL 10% Triton X-100
- 7.45 mL ddH2O
Procedure
NOTE: Do NOT use pipetting for resuspension—it can cause shearing of the genomic DNA.
Lysis
- Prepare all buffers and stock solutions beforehand and label all tubes
- All buffers (except Buffer QF and the 70% ethanol) should be equilibrated to room temperature
- Pre-warm the Buffer QF to 50°C to increase yields
- 70% ethanol should be cold (4°C)
- Grow S. meliloti cultures in 4-mL LB overnight to saturation
- Centrifuge two 1.5-mL aliquots of each culture at 13,200 rpm for 2 min. in 1.7-mL Eppendorf tubes
- Discard supernatant
- Thoroughly resuspend each pellet in 1 mL LB
- Centrifuge at 13,200 rpm for 2 minutes
- Discard supernatant
- Resuspend each pellet in 735 μL T10E1
- Add 39 μL 10% SDS to each tube
- Invert several times
- Immediately add 5 μL Proteinase K to each tube
- Vortex 5 seconds
- Incubate on the rotator at 37°C for 60 minutes
- Optional: Remove 300 μL and save for an analytical gel (test aliquot 1 = nuclear lysate)
CTAB Genomic DNA Isolation
- Add 125 μL 5 M NaCl to each tube
- Vortex 10 seconds
- Add 100 μL 10% CTAB to each tube
- Make sure the CTAB hasn't crashed out of solution; if it has, alternate vortexing and 10-minute incubations in the 60°C water bath until it is dissolved
- At this point a precipitate should form
- Vortex 10 seconds
- Incubate on the 60°C for 15 minutes
- Add 500 μL chloroform
- Vortex 10 seconds and shake to make a milky emulsion
- Centrifuge for 20 minutes at maximum speed
- A well-packed interface should form
- Using a P200, remove ~800 μL of the aqueous layer (top layer) to a new tube
- Do this slowly so that you don't suck up the debris at the interface
- Add 2 μL RNase A
- Vortex 5 seconds
- Incubate at 37°C for 20 minutes
- Optional: Remove 8 μL and save for an analytical gel (test aliquot 2 = CTAB purification)
Qiagen Genomic Column
- Suspend a QIAGEN Genomic-tip 20/G over a culture tube using a yellow tip holder
- Equilibrate the tip with 2 mL Buffer QBT and allow it to empty by gravity flow
- Add 200 μL Binding Buffer to each 1.7-mL tube
- Combine samples from the same original culture
- Be careful not to mix samples from different cultures!
- Add each sample to a tip and allow it to enter the resin by gravity flow
- You may have to apply positive pressure by mouth (1 drop every 5–10 seconds)
- Optional: Remove 300 μL and save for an analytical gel (test aliquot 3 = flow-through fraction)
- Move the tip and tip holder to a new culture tube
- Wash with 1 mL of Buffer QC and allow it to move through by gravity flow
- You may have to apply positive pressure by mouth (1 drop every 5–10 seconds)
- Wash again with 2 mL of Buffer QC and allow it to move through by gravity flow
- You may have to apply positive pressure by mouth (1 drop every 5–10 seconds)
- Optional: Remove 1.2 mL and save for an analytical gel (test aliquot 4 = wash fraction)
- Move the tip and tip holder to a new culture tube
- Elute with 2 mL of 50°C Buffer QF and allow it to move through by gravity flow
- You may have to apply positive pressure by mouth (1 drop every 5–10 seconds)
- Separate the eluate into 2 1.5-mL tubes (1-mL per tube)
- Precipitate the DNA with 700 μL of isopropanol
- At this point and on, you should include your test aliquots if you chose to collect them
- Rock gently for 1 minute
- Place tubes on ice for 15 minutes
- The DNA precipitate should sink to the bottom of the tube
- Centrifuge at maximum speed for 5 minutes
- Remove most of the isopropanol
- Centrifuge at maximum speed for 1 minute
- Remove the rest of the isopropanol with a P-200
- Add 100 μL cold 70% ethanol
- Do not pipette up and down or vortex!
- Centrifuge at maximum speed for 2 minutes
- Remove all of the ethanol with a P-200
- Add 60 μL of T10E1
- Drizzle the TE buffer twice down the back of the tube
- Do not pipette up and down or vortex!
- To dissolve the DNA, incubate at 60°C and flick once every minute for 15 minutes
- Combine DNA samples from the same original culture into one tube
- Final volume should be 120 μL
Analysis
- Analyze by gel electrophoresis on a 1% gel for 30 minutes at 120 V
- For test aliquots, add 2 μL 8X DNA loading dye to 2 μL DNA and load 4 μL
- For final DNA samples, perform a dilution series using 6 μL 8X DNA loading dye to 3 μL DNA (or 3 μL of the previous dilution) and load 4 μL
- The final concentration should be at least 5 ng in no more than 100 μL
Adapted from the QIAGEN Genomic DNA handbook and various CTAB procedures: