Plasmid DNA with Glass Beads & Glass Fibre Membrane

Materials

Glass beads: 212–150 µm size-fractionated, acid-washed glass beads from Sigma (G1145); or 710 to 1180 µm (cat. no. G1152)

Glass beads: EM Science or as <106 µm size-fractionated, acid-washed glass beads. 1.2 µm GF/C filters Whatman, ground GF/C material Whatman

Glass fiber: Ahlstrom 141, 142, Whatman GF/C, GF/D, Pall BioDyne, Whatman GMF 934AH (best, sigma Z690805-100EA), GMF 150 2 um, Pallfex 2500 AE 53009M, Ciro 23 um glass fiber filter

WHATMAN 7700-2801 GF/C is good for plasmid

http://www.whatman.com/GlassMicrofiberBinderFree.aspx

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC101856/

Glass beads were purchased as 212–150 µm size-fractionated, acid-washed glass beads from Sigma (St Louis, MO) or EM Science or as <106 µm size-fractionated, acid-washed glass beads. The 96-well filter used to retain the glass beads during the plasmid binding and washing steps was a Millipore 1.2 µm hydrophilic Durapore PVDF plate (Bedford, MA). For comparison, a 96-well Millipore 1.2 µm glass fiber type C and ground Whatman GF/C filter paper were used for 2-propanol binding experiments. Ground GF/C powder was prepared by grinding GF/C paper using a mortar and pestle, size fractionating the powder by three sedimentations and drying at 65°C for 2 h. Sedimentation was performed by resuspending the ground GF/C in 2-propanol, allowing the glass fiber to settle for 5 min and decanting the liquid suspension. To simplify the addition of a predetermined amount of glass beads to the Millipore filter, a predrilled 96-well plexiglass plate was constructed to reproducibly deliver ~80 mg of 212–150 µm size-fractionated glass beads to the filter in a high throughput manner.

The binding efficiency of the direct lysis protocol was further characterized by examining different glass materials, including different sized acid-washed glass beads (common silicate glass) and intact and ground glass fiber filters type C (GF/C) (borosilicate glass) (Fig. ​(Fig.1).1). To normalize the different glass materials, the amounts of glass beads and ground GF/C were measured using a fixed volume plexiglass dispenser (see Materials and Methods). We found that the direct lysis method coupled with 2-propanol precipitation gave high quality plasmid DNA in good yield using all the glass supports examined in Figure ​Figure11 (lanes 4–11). Typically, significantly lower yields of plasmid DNA were recovered when binding of the direct lysate was facilitated in a 1.25 M NaCl, 10% PEG solution (final concentration) (lanes 12 and 13). Performance of the direct lysis/2-propanol preparation method using the different glass materials, however, differed with respect to performance and recovery of reproducible quantities of plasmid DNA. In general, the 1.2 µm GF/C filters gave greater flow resistance during filtering of the direct lysate and the subsequent washing steps. Furthermore, the ground GF/C material gave variable recovery of plasmid DNA volumes eluted (lanes 8 and 9).For routine usage we found that 212–150 µm size-fractionated, acid-washed glass beads gave the most reliable results regarding plasmid DNA yield and protocol performance. The average plasmid yield was 21 ± 12 µg/ml of culture (x = 192).

Comparison of different glass supports and precipitation solutions for rat whole genome shotgun clones: lane 1, 500 ng pGEM plasmid template control; lanes 2 and 3, experiment A, ethanol precipitation of standard cleared alkaline lysates; lanes 4–11, 2-propanol precipitation: lanes 4 and 5, experiment B, using 212–150 µm acid-washed glass beads; lanes 6 and 7, experiment C, using <106 µm size-fractionated, acid-washed glass beads; lanes 8 and 9, experiment D, using a ground glass fiber filter (Whatman GF/C); lanes 10 and 11, experimentE, using a Millipore 1.2 µm glass fiber type C plate; lanes 12–13, experiment F, precipitation by 1.25 M NaCl, 10% PEG solution using 212–150 µm acid-washed glass beads. For each experiment 8.3% of the eluted product (5 µl) was loaded for the different glass support and precipitation solution variables prepared from 0.4 ml of bacterial cultures.

Filters

pre-filters:

Porex: Porex X-4920 10-20 um (PE)

Handee: Handee spin column frit (89869, HH106259) MN frit: Macherey Nagel QUICKPURE® column PE frit (Cat 406184)

depth filters:

113: Pall Absorbent Paper 113--P/N S70007, lot 20410

133: Pall Absorbent Paper 133--P/N S70008, lot 64515

165: Pall Absorbent Paper 165--P/N S70009, lot 70301

GFB: Whatman GF/B glass microfibre depth filter

For each pre-filter/depth filter combination (or control experiment), at least two parallel experiments were run. In general, the modified system generated plasmid DNA of comparable quality to those isolated using the control kits. Flocculants were retained on top of the pre-filters, yet the disposable columns all exhibited good flow characteristics. Minor quantities of KDS were observed in the flow through of the pre-filter columns containing Pall absorbent paper depth filters, but not in the pre-filter columns with the Whatman GF/B glass microfibre depth filter. This slightly higher salt levels in some of the minipreps, however, does not appear to be a problem for downstream applications as evidence by subsequent analysis of the isolated plasmid DNA.

Low levels of protein contaminants was observed. The amount of particulates in the final elution was equivalent to control extractions. The miniprep performed with pre-filter columns with the Pall absorbent paper depth filter has a higher yield in general, as compared to the minipreps using a pre-filter column with the Whatman GF/B glass microfibre depth filter (FIG. 3). This was expected, as the Whatman GF/B glass microfibre depth filter used could bind plasmid DNA. Majority of uncut plasmid DNA was in the supercoiled configuration (FIG. 4), although some genomic DNA was observed in the minipreps isolated with the pre-column containing the Pall absorbent paper depth filter. Restriction digest of the plasmid DNA was successful for all the samples isolated (FIG. 5), proving the quality of the plasmid DNA. Therefore in general the quality of the isolated plasmid DNA was comparable to control extractions.

Glass syringe filters:

National Scientific Co. (F2500-18) (Quakertown, PA): 30 mm glass syringe filters with a 0.7 um pore-size

Pall Life Science (4523T) (East Hills, NY): 25 mm glass syringe filters with a 1 um pore-size.

Millipore (APFF02500) (Billerica, MA): 25 mm glass filter discs with a 0.7 um pore-size

Sterlitech (540100) (Kent, WA): filter holder

4 glass syringe filters can purify up to 600 ug of plasmid from a cleared cell lysate (from 200 ml culture).  If equal volume of ice-cold isopropyl alcohol and 0.15 volumes of 3 M sodium acetate (pH 5.1) is added to lysate, followed by a 2 to 5 minute incubation on ice, the yield can be increased to 900 ug

Growth medium

TB

12 g tryptone

24 g yeast extract,

4 ml glycerol, water to 900 ml, autoclave

Then add  20 mL filter sterilized 1 M KH2PO4 and 80 mL filter sterilized 1 M K2HPO4

2XYT

10 g yeast extract
16 g tryptone (or peptone)
5g NaCl
4 ml glycerol, water to 900 ml, autoclave

Then add 20 mL filter sterilized 1 M KH2PO4 and 80 mL filter sterilized 1 M K2HPO4

LB

5g yeast extract
10 g tryptone (or peptone)
10 g NaCl
4 ml glycerol, water to 900 ml, autoclave

Then add 20 mL filter sterilized 1 M KH2PO4 and 80 mL filter sterilized 1 M K2HPO4

Grow E coli in one of the above media

Solutions

P1: Lysis Buffer (0.5 % (w/v) SDS, 8 % (w/v) PVP-10, 250 mM sodium chloride, 25 mM Na2-
EDTA, 200 mM Tris-HCl pH 7.5. RNase A (0.25 mg·mL-1) is added before use.

P1_1: 50 mM Glucose, 10 mM Tris, pH 7.5, 1mM EDTA, pH 8, 90 μg/ml RNase A
P2: 0.2 N NaOH, 1% SDS

P3-1: Precipitation Buffer (5 M potassium acetate, pH 6.5 adjusted with acetic acid).

P3_2: Binding Buffer (2 M Guanidinhydrochloride, 75 % (v/v) Ethanol).

P4: 4 volumes of 5 M potassium acetate, 1 volume of 10 M (57% v/v) glacial acetic acid
6 M potassium iodide

Use for Corning 0.66 mm glass fiber plate (Cat. No. 3511)  plate: 50 ul P1_1, 100 ul P2, 150 ul P4, 380 μL 6 M potassium iodide, 2X 200 μL of 80% ethanol to wash, 80 μL of 10 mM Tris, pH 8.5 to elute

PE: Washing Buffer I (10 mM NaCl, 10 mM Tris-HCl pH 6.5, 80 % (v/v) Ethanol).

Washing Buffer II (96 % Ethanol).

TE: Elution Buffer (10 mM Tris-HCl pH 8.5).

Low MW DNA binding solution: 8.5% PEG 8000/850 mM NaCl, add to cleared lysate

Make spin column with silicar membrane

Cut Fresh silica membranes  from GF/F borosilicate glass fiber paper (Whatman) using a 8 mm paper punch (or for single columns a 7 mm paper punch was used). Two layers of silica membranes are placed in each column and fixed in place with the gasket ring. If larger yields of DNA are required, a third membrane is added and fixed.

Results with silica membrane

6/24/2013. Test different type of columns. Elute with 50 ul EB

	Column 1	Column 2	Column 3	Home-made column 934-AH
Price	$0.12	$0.09	0.12
Low copy plasmid,pET30-Pfu, from 5 ml LB culture
1st Elute,  DNA conc, ng/ul	74.7	90.3	46.2	60.6
2nd Elute	19.6	19.8	17.2	20.3
High copy plasmid, pEC-Pfu, from 5 ml LB culture
1st Elute, DNA conc, ng/ul	255.1	193.8	292.8	79.1
2nd Elute	52.5	31.2	35.3	0?

Home-made columns using two differnt types of membrane

	10% NaOH soaking	0.1N HCl Washing
Whatman Membrane 934-AH
DNA conc	203.2	176.7
OD260/280	1.94	1.95
Whatman Membrane GF/F
DNA conc	118.3	240.2
OD260/280	1.95	1.88

Regeneration of Glass Beads column:

1. Rinse with 1 % detergent (any commercial dishwashing liquid) and water to remove cell debris.

2. Wash and incubate with  0.5 M HCl for 30 min to eliminate remaining DNA.

3. Rinse again with 1 % detergent followed by sterile water.

MaxiPrep

1. Grow 1 L E coli in LB for 18 hours at 37°C with shaking.

2. Spin down

3. Resuspend with 10 ml (or 25 ml) of ice-cold solution I (50 mM Tris.HCl pH 8.0, 10 mM EDTA pH 8.0, 100 ug/ml RNase A)

4. Lyse with 10 ml (or 25 ml) of solution II (0.2 M NaOH, 1% SDS)

5. Neutralize with 10 ml (or 25 ml) of solution III (3 M potassium acetate (Sigma) pH 5.3 with acetic acid)

6. Spin down at 6000 rpm for 5 min, filter through a coffee paper.

7. Add a half volume of ice-cold 6 M guanidine hydrochloride in 10% acetic acid to final conc of guanidine hydrochloride is 2 M)

This step can be omitted if N3 is used at step 5

8. Add 10% Triton X-114 to final conc of 0.5 %, incubate on ice for 10 min.

9. Pour to  a 50 ml syringe attached to syringe filter with 4 layers of 25 mm glass fiber membrane, or use peristaltic pump to load to membrane filter.

10. Wash two times with 10 ml PB.

11. Wash twice with 10 ml PE buffer 2 mM Tris.HCl pH 7.5, 20 mM NaCl in 80% EtOH

12. Elute DNA with 2 X 2 ml of TE (pH 8.0) or EB  for 2 minutes by pressing the plunger.

13. Add DNA isopropanol/sodium acetate (2 ml of 3 M sodium acetate (pH 5.1) and 15 ml of ice-cold isopropyl alcohol).

14. Spin 6000 rpm for 15 min at 4 oC.

15. Wash The pellet with 70% ethanol

16. Resuspend DNA with 1 ml of TE (pH 8.0) or entotoxin-free EB

Plate format

Standard U-bottom 96-well microplate with a 350-il-well volume (Costar, Corning, NY).

1. Add 45 µl of solution I to cells,  followed by shaking using an orbital shaker at full speed for 10 min to facilitate cell lysis.

2. Add 45 µl of solution II, shake for 1 min, stand for 2 min.

3. Add 45 µl of solution III, shake for 2 min.

4.  Spin and transfer supernatant to glass bead filter plate.

5. Dispense 205 µl of 2-propanol  into the glass bead filter plate using a Q-fill2 pipettor (Genetix).

6. Wait for 5 min (i.e. no mixing) to facilitate binding of the 2-propanol-precipitated plasmid DNA to the glass beads,

7. Apply vacuum pressure  using a Millipore vacuum manifold to remove the cell lysate solution. 8. Was glass bead filters  eight times with 200 µl of 80% aqueous ethanol by vacuum filtration.  9. Dry filter plates using a SpeedVac under heat.

10. Add  60 µl of 10 mM Tris–HCl, pH 8.0, 0.2 mM EDTA containing 0.16 mg/ml RNase A, stand for 1 min

11. Elute DNA by vacuum filtration into a 96-well collection plate.

Detergents for removal of endotoxin

		Average	Average
		Plasmid	Endotoxin
		Yield	Levels
Detergent Class	Detergent	(ug)	(EU/mg)
	No Detergent Control	29	>4500
Alkyl	Detergent control	22	32
thioglucoside	5% n-octyl-β-D-1-
	thioglucopyranoside
Alkyl glucoside	2.5% n-octyl-β-D-	37	120
	glucopyranoside
	5% n-octyl-β-D-	29	26
	glucopyranoside
Alkyl maltoside	10% n-decyl-p-D-	16	58
	maltopyranoside
Alkyl sulfate	0.5% sodium dodecyl	42	*
	sulfate
	1.0% sodium dodecyl	46	1800
	sulfate
Zwitterionic	0.5% SB3-14	6	*
	1.0% SB3-14	2	1700
	2.5% SB3-14	1	*
	5.0% SB3-14	2	*
	10.0% SB3-14	1	*
CHAPS series	2.5% CHAPS	13	>7300
	5.0% CHAPS	6	>14000
	10.0% CHAPS	1	*
	2.5% CHAPSO	11	>8200
	5.0% CHAPSO	7	>13000
	10.0% CHAPSO	2	*
Glucamide	10.0% MEGA-8	9	6000
Polyoxyethylenes	0.5% TRITON X-100	7	*
	1.0% TRITON X-100	4	*
	2.5% TRITON X-100	2	4700
	2.5% TRITON X-114	2	6400
	5.0% TRITON X-114	2	*
	2.5% TWEEN 20	2	>6300
	5.0% TWEEN 20	2	*
	1.5% BRIJ 35	1	>11000
	3.0% BRIJ 35	1	*
* Not tested.