Excellgen at 19:29, 11 July 2014

2014-07-11T19:29:46Z

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== Qiagen or MN Protocol ==

Use Qiagen tip 2500 (Mega) or MN AX 2000 (Mega), or AX 10000 (Giga) columns. 

Follow their protocol to elute plasmid DNA from column. 

For Qiagen column add 0.1% Triton X-114 to cleared lysate and incubate on ice for 20 min before loading to column.

For Qiagen column elute plasmid with QF buffer. 

'''QF buffer '''(elution buffer)

1.25M NaCl 50 mM Tris-HCl pH 8.5 15% isopropanol

to make 2 L 833 ml 3M NaCl 100 ml 1M Tris pH 8.5 300 ml EtOH dH2O to 2 liters check the pH and adjust to 8.5 (it should be close) with NaOH or HCl store QC and QF in tightly capped containers at room temp. 

== Optional: Endotoxin and RNA reduction Protocol ==

1. Spin down cells from 1 L LB culture for 5 min at 6000 rpm 2. Resuspend pellet in 50 ml of cold 20% sucrose , 50 mM Tris-HCl, pH 8.0 . 3. Add 10 ml of freshly prepared lysozyme (5 mg/ml in 0.25 M Tris, pH 8.0) 4. Add 20 ml of cold 0.25M EDTA, pH 8. 5. Leave on ice for 5 min. 6. Add 20 ml of cold 0.25M Tris-HCl, pH 8.0, incubate at 37 oC for  30 min. 7. Spin at 4000 g for 10 min, remove viscous supernatant carefully. 8. Resuspend spheroplast pellet in 120 ml of 50 mM glucose/25 mM Tris-HCl, pH 8.0/10 mM EDTA, pH 8.0 containing 100 ug/ml RNase A, 

or P1 containing purified RNAse (DNase-free RNase) or heat-treated RNase. 

or 120 ml S1-EF 

 9. Incubate at 37 oC for 30 min or room temperature o/n. 

 10. Add 120 ml of P2 (or S2-EF) to lyse the cells. 11. Add 120  ml of P3 (or S3-EF). 12. Spin 6000 X RPM 10 min 13. Collect cleared lysate, filter through a coffer paper. 

== Plasmid Capture using MN NucleoBond AX 10000 Giga column. ==

1. Regenerate one  MACHEREY-NAGEL NucleoBond AX 10000 (Giga) column using 150 ml 2 M NaCl. 

if flow rate is very slow, wash with 50 ml 0.1N NaOH then 200 ml water before regenerating using 150  ml of 2 M NaCl

2. Equilibrate column with 100 mL Buffer N2. 

3. Load cleared lysate onto the column 

4. Wash with 4 x 150 ml of N3-EF. 

5. Wash with 3 X 130 ml of  N4-EF. 

6. Elute with 100 mL of N5-EF. 

 

== Purify Plasmid DNA from Isopropanol Precipitate ==

1. If Qiagen anion exchange column is not available, use 0.6 vol isopropanol to precipitate plasmid DNA from lysate (P1 + P2 + P3). 

Alternatively, DNA will absorb to acid washed autoclaved SiO2 particles when the lysate is mixed with 15%~60% isopropanol, 0.1% CTB after incubating for 30 min. Spin down SiO2 particles and wash 2 times with PE, then elute plasmid DNA  with 2 to 3 times of 1.5 ml hot (80 oC) TE/EB. Before loading into CHT Ceramic Hydroxyapatite column, the DNA solution shold be mixed with 9 vol of 7~8 M urea, 0.24 M sodium phosphate buffer pH 6.8, 0.1% SDS or CTAB 

2. Add 0.6 vol of isopropanol, incubate at RT for 10 ~15 min, spin at 5000 rpm for 10~15 min, rinse pellet once with 70% EtOH to remove as much of acetate as possible. 

3. Dissolve pellet in 10 ml '''Binding Buffer: '''8 M urea, 0.24 M sodium phosphate buffer pH 6.8~7.0, '''0.1% SDS or CTAB'''. 

4. Mix solubilized solution with 1 g  Bio-Rad CHT Ceramic Hydroxyapatite Type II Media (1 g powder will be enough to purify plasmid DNA from 1 L culture). Incubate at RT for 15 min with ocassional mixing. 

5. Spin and decant supernatant carefully.  Wash the particles with 10 ml of '''Washing Buffer 1''': 8 M urea, 0.24 M phosphate buffer, pH 6.8. Spin down particles, if necessary wash with Washing Buffer 1  until the absorbance at 260 nm of supernatant is approximately zero. 

6. Wash with ~45 ml of '''Washing Buffer 2''': 10 mM phosphate buffer, pH 6.8 (to remove urea). Spin and remove supernatant. 

7. Add ~10 ml Washing Buffer 2 and transfer particles to a Pierce spin column.

8. Spin 3000 rpm for 5 min to remove residual washing buffer.

9. Add 1 ml  '''Elution Buffer''': 0.3  M phosphate buffer, pH 6.8, soak 1 min, spin at 3000 rpm for 5 min and collect 1  ml fractions, continue to elute until OD260 ~> 0.05. Total elution vol is about 10 ml for 1 g particles. 

10. Combine fractions containing DNA. Measure OD260.

11. Dilute DNA solution with equal volume of water, add 0.6 vol isopropanol to precipitate DNA onto a "Qiagen" precipitator (filter) 

12. Elute DNA with TE or EB. 

13. Measure DNA conc using nanodrop 

14. Run gel to check purity (to see if there is any RNA or genomic DNA contaimination) 

15. Regenerate CaPO4 particles with  1 N NaOH, then 1 M NaPO4 buffer, pH 7.0, wash with water. Store in 20% ethanol.

'''7/9/14''' 

'''Binding of plasmid to  HTP particles''' 

[[Image:Hydroxylapatide plasmid 7 9 14 2.JPG]] 

{| width="496" cellspacing="1" cellpadding="1" border="1"
|-
| 
| 1 
| 2 
| 3 
| 4 
| 5 
| 6 
| 7 
|-
| HTP types 
| Bio-Rad HTP 
| Bio-Rad HTP
| Bio-Rad Ceramic HTP type 1  
| Bio-Rad Ceramic HTP type 1
| Bio-Rad HTP DNA Grade 
| Bio-Rad HTP DNA Grade
| Plasmid DNA pellet solubilized in Binding Buffer 
|-
| Phosphate Conc. 
| 0.24M NaPO4 
| 0.12M NaPO4
| 0.24M NaPO4
| 0.12M NaPO4
| 0.24M NaPO4
| 0.12M NaPO4
| 
|}

Bio-Rad Ceramic HTP type 1 does not bind plasmid DNA very well 

'''Plasmid Eluted from HTP particles''' 

[[Image:Hydroxylapatide plasmid 7 9 14.JPG]] 

{| width="436" cellspacing="1" cellpadding="1" border="1"
|-
| 1 
| 2 
| 3 
| 4 
|-
| Bio-Rad HTP
| Bio-Rad Ceramic HTP type 1
| Bio-Rad HTP DNA Grade
| Plasmid DNA pellet solubilized in Binding Buffer 
|}

7/11/14 

maxiprep 

[[Image:CaPO4 plasmid 7 11 14.JPG]] 

{| width="492" cellspacing="1" cellpadding="1" border="1"
|-
| 1
| 2
| 3
| 4
|-
| Bio-Rad HTP 
| Bio-Rad HTP DNA Grade 
| Bio-Rad HTP 
| Bio-Rad HTP DNA Grade 
|-
| Clone 470 
| 470 
| 471 
| 471 
|}

 

 

'''For bioling plasmid protocol:''' 

Resuspend E coli in 10 mM Tris, 10 mM NaCI, 2 mM EDTA, pH 7.6. Heat the suspension at 100 "C for 2 min, then rapidly freezing to - 70 "C in dry-ice /EtOH bath. Warm up and spin to remove pellet. Mix supernatant with 9 vol of 7~8 M urea, 0.24 M sodium phosphate buffer pH 6.8, 0.1% SDS or CTAB. 

 

'''For lysome protocol:''' 

For 1 L culture 

Resuspend E coli in 4 ml of 0.25 M sucrose, 2 mM MgC12 

Add 0.8 ml (1/5 volume) of lysozyme solution  20 mg/ml lysozyme (Sigma Ltd) in 10 mM Tris/HCl, pH 8.0. 

Incubate for 5 min on ice 

Add 1.6 ml (2/5 volume of inital volume) of  0.25 M EDTA, pH 8.0 

Incubate for 5 min on ice 

Add 6.4 ml (equal vol) of  0.5 % (w/v) Triton X-100, 62.5 mM EDTA, 50 mM Tris-HCl, pH 8.0 

Incubate for 20 rnin on ice, swirl every 5 min. 

Spin and collect supernatant, mix cleared solution with 9 vol of 7~8 M urea, 0.24 M sodium phosphate buffer pH 6.8, 0.1% SDS or CTAB. 

 

=== Regeneration and Storage of Bio-Rad CHT Ceramic Hydroxyapatite Type II. ===

 0.5M sodium phosphate, pH 7 1 M trisodium phosphate, pH 11–12 

 Autoclave: 121°C, 20 min in phosphate buffer, pH 7 

Sanitization: 1–2 N NaOH Recommended column storage: 0.1 M NaOH 

== Plasmid Purification using hydroxyapatite ==

Use 1 g hydroxyapatite for 1 litre of bacterial culture. This will give 3~5 ml wet beads.

1. Weigh 2X 0.5 g hydroxyapatite powder (Biorad, DNA grade, will bind 500 to 800 ug DNA, Cat #130-0520, or 130-0420, non-DNA grade), add to two 50 ml tubes. Add 30 ml water. Let the beads settle for 10 min. Decant to remove fine particles.

2. Resuspend hydroxyapatite beads in 5 ml  (10  column volumes)  of '''buffer A''' 10 mM NaPO , 1 mM EDTA, pH 7.0 sodium phosphate buffer. Let beads settle for 3 min. Carefully decant the liquid and fine particles.

 

 

3. <strike></strike>Add 50 ml of N5-EF eluate (should contain at least 500 mM NaCl) from AX 10000 column or Qiagen tip 2500 to each 50 ml tube containing HA beads. 

Cap the tubes and tighten the caps.

Put onto a rotating mixe, mix at room temperature,  let plasmid DNA bind to the beads for 10 to 30 min. 

4. Spin down beads, use  '''buffer A''' to transfer beads to one Pierce 10 ml spin column.

 

'''buffer A ''' 

 

5. Spin at 1000 rpm for 1 min to remove residual buffer. 

 

6. Elute plasmid DNA with 10 X 1 ml of  '''0.4 M '''phosphate buffer, pH 6.8~ 7.0, collect 1 ml fractions: 

 

*6.1 Add 1 ml, soak for 1~2 min, spin at 1000 rpm for 30 sec.
*6.2 Add 0.5 or 1 ml, soak for 1~2 min, spin at 1000 rpm for 30 sec.
*6.3 repeat the above elution 6.2 step, check OD260 using nanodrop until OD260 < 50 ug/ul.

 

7. Use nanodrop to measure DNA conceration, OD260/280 ratio, and check purity by agarose gel electrophoresis. 

8. Combine fractions containing plasmid DNA, filter through a 0.22 micron filter in the biosafty hood. label tubes and store them at -20 oC. 

9. Combine used beads and store in cold room. Beads can be regenerated using 1M Phosphate Solution '''for protein purification''' only. 

10/14/2013 

Plasmid DNA purified using hydroxyapatite beads. 0.5 ml per fraction, load 10 ul to gel, concentrations: 

38, 322, 333, 227, 171, 168, 126 ng/ul

[[Image:Plasmid hydroxyapatide.JPG]] 

 

10/16/2013

Plasmid DNA purified using hydroxyapatite beads. 0.5 ml per fraction, load 600ng to gel, concentrations:

170, 209, 218, 305, 642, ng/ul ,OD260/280 ratio:~1.9 

[[Image:Plasmid QC 10-16-2013.JPG]] 

 

 

 

== Buffers ==

'''1M Phosphate Solution, pH 6.8''' 

In a 1L flask dissolve 119.98g of sodium phosphate monobasic in 1L of  ddH2O. 

Prepare a 1M sodium phosphate dibasic solution in a 1L flask by dissolving 141.96g of sodium phosphate dibasic in 1L ddH2O. 

Combine the mono- and di- solutions at a ratio of 1:1. 

Optional: adjust pH to 6.8 by adding sodium hydroxide (to increase pH) or phosphoric acid (to decrease pH). 

'''10 mM Phosphate Buffer''' 

 

10 ml of 1M Phosphate Solution, pH 6.8, 

250 ml of 4 M NaCl

2 ml of 0.5M EDTA. 

Add ddH2O to a volume of 1000 ml. 

Autoclave. 

 

 '''0.4 M Phosphate Buffer''' 

400ml of 1M Phosphate Solution, pH 6.8~7.0, 

1 ml of 0.5M EDTA. 

ddH2O to 500 ml 

Autoclave.

 

'''Plasmid Purification Buffers for AX10000 column''' 

{|
|-
| bgcolor="#ffffcc" | 
| bgcolor="#ffffcc" | Buffer Name 
| bgcolor="#ffffcc" | Recipe 
| bgcolor="#ffffcc" | Buffer Name
| bgcolor="#ffffcc" | Recipe
| bgcolor="#ffffcc" | Buffer Name
| bgcolor="#ffffcc" | Recipe 
| bgcolor="#ffffcc" | Buffer Name
| bgcolor="#ffffcc" | Recipe
|-
| bgcolor="#ffffcc" | 
| bgcolor="#ffffcc" |   '''N2-EF'''
| bgcolor="#ffffcc" | 1 L
| bgcolor="#ffffcc" | '''N3-EF'''
| bgcolor="#ffffcc" | 1 L
| bgcolor="#ffffcc" | '''N4-EF''' 
| bgcolor="#ffffcc" | 1L 
| bgcolor="#ffffcc" | '''N5-EF'''
| bgcolor="#ffffcc" | 1L
|-
| bgcolor="#ffffcc" | 
| bgcolor="#ffffcc" | 
| bgcolor="#ffffcc" | 12.1 g
| bgcolor="#ffffcc" | 100 mM Tris
| bgcolor="#ffffcc" | 12.1 g
| bgcolor="#ffffcc" | 100 mM Tris 
| bgcolor="#ffffcc" | 12.1 g 
| bgcolor="#ffffcc" | 100 mM Tris 
| bgcolor="#ffffcc" | 12.1 g 
|-
| bgcolor="#ffffcc" | 
| bgcolor="#ffffcc" | 15 % EtOH
| bgcolor="#ffffcc" | 150 ml
| bgcolor="#ffffcc" | 15 % EtOH
| bgcolor="#ffffcc" | 150 ml
| bgcolor="#ffffcc" | <strike></strike>15 % EtOH<strike> </strike>
| bgcolor="#ffffcc" | 150 ml 
| bgcolor="#ffffcc" | 15 % EtOH 
| bgcolor="#ffffcc" | 150 ml 
|-
| bgcolor="#ffffcc" | 
| bgcolor="#ffffcc" | 900 mM KCl
| bgcolor="#ffffcc" | 67.1 g
| bgcolor="#ffffcc" | 1.15 M KCl
| bgcolor="#ffffcc" | 85.7 g
| bgcolor="#ffffcc" | 1.15 M KCl 
| bgcolor="#ffffcc" | 85.73 g 
| bgcolor="#ffffcc" | 1 M KCl
| bgcolor="#ffffcc" | 74.56 g
|-
| bgcolor="#ffffcc" | 
| bgcolor="#ffffcc" | 0.15 % TX 100
| bgcolor="#ffffcc" | 1.5 g Triton X-100 
| bgcolor="#ffffcc" | 0.15 % TX 100
| bgcolor="#ffffcc" | 1.5 g Triton X-100 
| bgcolor="#ffffcc" | 
| bgcolor="#ffffcc" | 
| bgcolor="#ffffcc" | 
| bgcolor="#ffffcc" | 
|-
| bgcolor="#ffffcc" | 
| bgcolor="#ffffcc" | 
| bgcolor="#ffffcc" | pH '''6.3''' with H3PO4 
| bgcolor="#ffffcc" | 
| bgcolor="#ffffcc" | pH '''6.3''' with H3PO4 
| bgcolor="#ffffcc" | 
| bgcolor="#ffffcc" | pH '''6.3''' with H3PO4 
| bgcolor="#ffffcc" | 
| bgcolor="#ffffcc" | pH 8.5 with H3PO4 
|}

 '''Hydroxyapatite hydration''': Weight out 1g of hydroxyapatite and add to a 50ml tube. Add 6ml of 10 mM phosphate buffer to the hydroxyapatite and invert to mix. Let beads settle down for 10 min and remoe fine particles by decanting. 

Store for extended periods of time at 4°C. 

 

 

 

'''Ref:''' 

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3230181/ 

= =

10/6/2013.

1 ml E coli lysate, precipitate with 3 M KCl, adjust to pH 4.0 after adding KCl to final conc 1M.

or precipitate with 3 M KoAc (P3).

Bind to 10 mg HA beads.

HA beads were partially dissolved by KoAc, only 50% of the beads is intact after overnight incubation.

Miniprep 

[[Image:HA plasmid KCl vs KAc.JPG]]

Lane 1: Precipitate with KCl

Lane 2: Precipitate with KoAc

[[Category:Protocols]]

Plasmid MaxiPrep Hydroxyapatite - Revision history

Excellgen at 19:29, 11 July 2014