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#314
Molecular Biology
Complete
Miniprep: pUC19-GFP from DH5α o/n (5 mL)
This template captures small-scale isolation of pUC19-GFP plasmid DNA from a 5 mL overnight culture of E. coli DH5α using the QIAprep Spin Miniprep Kit (silica column chemistry).
Goal: isolate 5-15 µg of pure pUC19-GFP plasmid suitable for restriction digest verification, Sanger sequencing, and downstream transfection or cloning steps.
Method overview: 5 mL of overnight LB+Amp culture is pelleted at 8,000 × g for 3 minutes, resuspended in 250 µL P1 buffer (with RNase A), lysed by adding 250 µL P2 alkaline lysis buffer, neutralized with 350 µL N3 buffer, cleared by centrifugation, applied to the QIAprep column, washed once with PB and twice with PE buffer (containing ethanol), dried, and eluted in 50 µL EB buffer pre-warmed to 70°C. Concentration confirmed on Qubit dsDNA HS; purity confirmed on NanoDrop.
Expected: 150-300 ng/µL eluate concentration (7-15 µg total yield from 5 mL DH5α culture with pUC19 high-copy vector), A260/A280 between 1.8 and 1.95, supercoiled-dominant band on diagnostic gel.
Last saved: 34 seconds ago
10 fields loaded from Plasmid Miniprep baseline (SOP v1.6)
Culture
Host strain
select
DH5α, TOP10, JM109, Stbl3, XL1-Blue
Plasmid
text
pUC19-GFP
Culture volume
number
5mL
Lysis
Buffer set
select
P1/P2/N3 (Qiagen), Cell Resuspension/Lysis/Neutralization (Promega)
RNase A in P1
select
yes, no
Neutralization temp
select
room temp, 4 °C
Elution & QC
Elution buffer
select
EB buffer, nuclease-free H₂O, TE
Elution volume
number
50µL
Concentration
number
218ng/µL
A260/A280
number
1.86
01
0:05
Pellet 5 mL o/n culture (8k × g × 3 min), decant supernatant
02
0:03
Resuspend pellet in 250 µL P1 buffer (with RNase A), vortex to homogeneity
03
0:05
Add 250 µL P2 lysis buffer, invert 6×, incubate 5 min RT (do NOT vortex)
04
0:02
Add 350 µL N3 neutralization buffer, invert 6×, white precipitate forms
05
0:12
Centrifuge 14k × g × 10 min, decant clear supernatant to QIAprep column
06
0:02
Spin column 14k × g × 1 min, discard flow-through
07
0:02
Wash with 500 µL PB buffer, spin, discard flow-through
08
0:04
Wash with 750 µL PE buffer, spin, discard, additional spin to dry membrane
09
0:05
Transfer column to clean 1.5 mL tube, add 50 µL EB (70°C), incubate 2 min, spin to elute
10
0:08
QC: 1 µL NanoDrop A₂₆₀/A₂₈₀ + 1 µL Qubit dsDNA HS, archive at -20°C
| A | B | C | D | E | F | G | |
|---|---|---|---|---|---|---|---|
| 1 | Colony | Vol (µL) | Conc (ng/µL) | A260/A280 | A260/A230 | Yield (µg) | Notes |
| 2 | C1 | 50 | 218 | 1.86 | 2.04 | 10.9 | Best yield |
| 3 | C2 | 50 | 174 | 1.84 | 1.96 | 8.7 | Within spec |
| 4 | C3 | 50 | 195 | 1.88 | 2.08 | 9.8 | Within spec |
| 5 | C4 | 50 | 142 | 1.82 | 1.92 | 7.1 | Lower yield — possible plasmid loss |
| 6 | C5 | 50 | 203 | 1.87 | 2.06 | 10.2 | Within spec |
| 7 | no-ins ctrl | 50 | <2 | na | na | na | Confirms no false plasmid in untransformed |
EXP-2026-0604
Colony pick from pUC19-GFP transformation plate
A. Marin
Jun 4
EXP-2026-0606
Diagnostic restriction digest + Sanger seq
A. Marin
Jun 6
SOP-0316
Plasmid Miniprep SOP v1.6 (QIAprep chemistry)
QA Team
Mar 12
PLM-0048
pUC19-GFP plasmid map + sequence
Plasmid DB
Sep 4
KIT-0119
Qiagen QIAprep Spin Miniprep Kit insert
Vendor Docs
Jan 14
Visibility
Marin LabA. Marin
Can write
A. MarinDr. J. Park
Use Ctrl + click to add text
{
"experiment": {
"id": 314,
"custom_id": "MP-pUC19-2026-06",
"title": "Miniprep: pUC19-GFP from DH5α o/n (5 mL)",
"template": "Plasmid Miniprep",
"template_version": "v3.2",
"category": "Molecular Biology",
"status": "Complete",
"started_on": "2026-07-15",
"tags": [
"miniprep",
"plasmid",
"alkaline-lysis",
"silica-column",
"pUC19"
]
},
"main_text": "This template captures small-scale isolation of pUC19-GFP plasmid DNA from a 5 mL overnight culture of E. coli DH5α using the QIAprep Spin Miniprep Kit (silica column chemistry).\nGoal: isolate 5-15 µg of pure pUC19-GFP plasmid suitable for restriction digest verification, Sanger sequencing, and downstream transfection or cloning steps.\nMethod overview: 5 mL of overnight LB+Amp culture is pelleted at 8,000 × g for 3 minutes, resuspended in 250 µL P1 buffer (with RNase A), lysed by adding 250 µL P2 alkaline lysis buffer, neutralized with 350 µL N3 buffer, cleared by centrifugation, applied to the QIAprep column, washed once with PB and twice with PE buffer (containing ethanol), dried, and eluted in 50 µL EB buffer pre-warmed to 70°C. Concentration confirmed on Qubit dsDNA HS; purity confirmed on NanoDrop.\nExpected: 150-300 ng/µL eluate concentration (7-15 µg total yield from 5 mL DH5α culture with pUC19 high-copy vector), A260/A280 between 1.8 and 1.95, supercoiled-dominant band on diagnostic gel.",
"extra_fields": {
"Culture": {
"Host strain": {
"type": "select",
"value": "DH5α, TOP10, JM109, Stbl3, XL1-Blue"
},
"Plasmid": {
"type": "text",
"value": "pUC19-GFP"
},
"Culture volume": {
"type": "number",
"value": "5 mL"
}
},
"Lysis": {
"Buffer set": {
"type": "select",
"value": "P1/P2/N3 (Qiagen), Cell Resuspension/Lysis/Neutralization (Promega)"
},
"RNase A in P1": {
"type": "select",
"value": "yes, no"
},
"Neutralization temp": {
"type": "select",
"value": "room temp, 4 °C"
}
},
"Elution & QC": {
"Elution buffer": {
"type": "select",
"value": "EB buffer, nuclease-free H₂O, TE"
},
"Elution volume": {
"type": "number",
"value": "50 µL"
},
"Concentration": {
"type": "number",
"value": "218 ng/µL"
},
"A260/A280": {
"type": "number",
"value": "1.86"
}
}
},
"attached_files": [
{
"filename": "pUC19-GFP_map.pdf",
"type": "pdf",
"size": "212 KB"
},
{
"filename": "nanodrop_readings.csv",
"type": "csv",
"size": "6 KB"
},
{
"filename": "diagnostic_digest.tif",
"type": "png",
"size": "1.8 MB"
}
],
"steps": [
{
"n": 1,
"description": "Pellet 5 mL o/n culture (8k × g × 3 min), decant supernatant",
"duration": "0:05",
"complete": true,
"note": "OD₆₀₀ at harvest 4.2"
},
{
"n": 2,
"description": "Resuspend pellet in 250 µL P1 buffer (with RNase A), vortex to homogeneity",
"duration": "0:03",
"complete": true
},
{
"n": 3,
"description": "Add 250 µL P2 lysis buffer, invert 6×, incubate 5 min RT (do NOT vortex)",
"duration": "0:05",
"complete": true,
"note": "Lysate clears + viscous"
},
{
"n": 4,
"description": "Add 350 µL N3 neutralization buffer, invert 6×, white precipitate forms",
"duration": "0:02",
"complete": true
},
{
"n": 5,
"description": "Centrifuge 14k × g × 10 min, decant clear supernatant to QIAprep column",
"duration": "0:12",
"complete": true
},
{
"n": 6,
"description": "Spin column 14k × g × 1 min, discard flow-through",
"duration": "0:02",
"complete": true
},
{
"n": 7,
"description": "Wash with 500 µL PB buffer, spin, discard flow-through",
"duration": "0:02",
"complete": true
},
{
"n": 8,
"description": "Wash with 750 µL PE buffer, spin, discard, additional spin to dry membrane",
"duration": "0:04",
"complete": true,
"note": "Dry membrane prevents ethanol carry-over"
},
{
"n": 9,
"description": "Transfer column to clean 1.5 mL tube, add 50 µL EB (70°C), incubate 2 min, spin to elute",
"duration": "0:05",
"complete": true
},
{
"n": 10,
"description": "QC: 1 µL NanoDrop A₂₆₀/A₂₈₀ + 1 µL Qubit dsDNA HS, archive at -20°C",
"duration": "0:08",
"complete": true,
"note": "Qubit 209 ng/µL · A₂₆₀/A₂₈₀ 1.86"
}
],
"spreadsheet": [
[
"Colony",
"Vol (µL)",
"Conc (ng/µL)",
"A260/A280",
"A260/A230",
"Yield (µg)",
"Notes"
],
[
"C1",
"50",
"218",
"1.86",
"2.04",
"10.9",
"Best yield"
],
[
"C2",
"50",
"174",
"1.84",
"1.96",
"8.7",
"Within spec"
],
[
"C3",
"50",
"195",
"1.88",
"2.08",
"9.8",
"Within spec"
],
[
"C4",
"50",
"142",
"1.82",
"1.92",
"7.1",
"Lower yield — possible plasmid loss"
],
[
"C5",
"50",
"203",
"1.87",
"2.06",
"10.2",
"Within spec"
],
[
"no-ins ctrl",
"50",
"<2",
"na",
"na",
"na",
"Confirms no false plasmid in untransformed"
]
],
"links": {
"experiments": [
{
"ref_id": "EXP-2026-0604",
"title": "Colony pick from pUC19-GFP transformation plate",
"owner": "A. Marin",
"date": "Jun 4"
},
{
"ref_id": "EXP-2026-0606",
"title": "Diagnostic restriction digest + Sanger seq",
"owner": "A. Marin",
"date": "Jun 6"
}
],
"resources": [
{
"ref_id": "SOP-0316",
"title": "Plasmid Miniprep SOP v1.6 (QIAprep chemistry)",
"owner": "QA Team",
"date": "Mar 12"
},
{
"ref_id": "PLM-0048",
"title": "pUC19-GFP plasmid map + sequence",
"owner": "Plasmid DB",
"date": "Sep 4"
},
{
"ref_id": "KIT-0119",
"title": "Qiagen QIAprep Spin Miniprep Kit insert",
"owner": "Vendor Docs",
"date": "Jan 14"
}
]
},
"compounds": [
{
"name": "QIAprep Spin Miniprep Kit (250 preps)",
"catalog_lot": "27106 · Lot 174218923",
"stock_status": "In stock"
},
{
"name": "P1 resuspension buffer (with RNase A added)",
"catalog_lot": "19051 · Lot 172419428",
"stock_status": "In stock"
},
{
"name": "P2 lysis buffer (NaOH / SDS)",
"catalog_lot": "19052 · Lot 172419428",
"stock_status": "In stock"
},
{
"name": "N3 neutralization buffer (KOAc / acetic acid)",
"catalog_lot": "19053 · Lot 172419428",
"stock_status": "In stock"
},
{
"name": "PB binding buffer (guanidinium-HCl)",
"catalog_lot": "19066 · Lot 172419428",
"stock_status": "In stock"
},
{
"name": "PE wash buffer (with EtOH added)",
"catalog_lot": "19065 · Lot 172419428",
"stock_status": "Low"
}
],
"storage": [
{
"name": "Plasmid stock (50 µL working aliquots)",
"location": "-20 °C · Freezer B-1 · Drawer 3 · Box MP-2026-06",
"count": "×6"
},
{
"name": "Glycerol stock backup (colony picks)",
"location": "-80 °C · Freezer A-3 · Drawer 4 · Box GS-pUC19",
"count": "×5"
},
{
"name": "Eluate archive (sequencing-grade, 10 µL)",
"location": "-20 °C · Freezer B-1 · Drawer 3 · Box MP-ARCH-06",
"count": "×6"
}
],
"permissions": {
"visibility": [
{
"type": "Team",
"name": "Marin Lab"
},
{
"type": "Person",
"name": "A. Marin"
}
],
"can_write": [
{
"type": "Person",
"name": "A. Marin"
},
{
"type": "Person",
"name": "Dr. J. Park"
}
]
}
}