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Troubleshooting Guide for DNA Cleanup and Plasmid Purification

Need some help with your DNA cleanup or plasmid purification? We’re here to help. Our troubleshooting guide below outlines some of the most common pain points that scientists encounter during DNA purification of fragments and plasmids. Still having trouble after reviewing this? Contact our technical support at any time.

PROBLEM PRODUCT CAUSE SOLUTION
No DNA purified

Monarch Plasmid Miniprep Kit (NEB #T1010)

Monarch Spin Plasmid Miniprep Kit (NEB #T1110)

 Buffers added incorrectly
  • Add buffers in the correct order so that the sample is bound, washed and eluted in the correct sequence.
  • Ensure ethanol was added to Plasmid Wash Buffer 2
Plasmid loss during culture growth
  • Ensure proper antibiotic and concentration was used to maintain selection during culture growth.
Monarch DNA Gel Extraction Kit (NEB #T1020) Ethanol not added to DNA Wash Buffer
  • Ensure the proper amount of ethanol was added to Monarch DNA Wash Buffer
Monarch PCR & DNA Cleanup Kit (NEB #T1030) Ethanol not added to DNA Wash Buffer
  • Ensure the proper amount of ethanol was added to Monarch DNA Wash Buffer
Low DNA yield

Monarch Plasmid Miniprep Kit (NEB #T1010)

Monarch Spin Plasmid Miniprep Kit (NEB #T1110)

 

 Incomplete lysis
  • Pellet must be completely resuspended before addition of Plasmid Lysis Buffer (B2) – color should change from light to dark pink.
  • Do not use too many cells to avoid overloading the column. If culture volume is larger than recommended, scale up buffers B1-B3.
Plasmid loss during culture growth
  • Ensure proper antibiotic and concentration was used to maintain selection during culture growth.
Low-copy plasmid selected
Lysis of cells during growth
  • Harvest culture during transition from logarithmic growth to stationary phase (~12–16 hours).
Incomplete neutralization
  • Invert tube several times until color changes to yellow.
Incomplete elution
  • Deliver Elution Buffer directly to center of column.
  • Larger elution volumes and longer incubation times can increase yield.
  • For elution of plasmids >10 kb, heat the DNA Elution Buffer to 50°C and extend incubation time to 5 minutes).
Monarch DNA Gel Extraction Kit (NEB #T1020) Reagents added incorrectly
  • Be sure that buffers have been reconstituted correctly, and that reagents have been added in the correct order.
Gel slice not fully dissolved
  • Undissolved agarose may clog the column and interfere with binding. Incubate in Monarch Gel Dissolving Buffer for proper time and temperature.
Gel dissolved above 60°C
  • Dissolve gel slice in specified range (37-55°C). Higher temperatures can denature DNA.
Incomplete elution during preparation
  • Deliver Elution Buffer directly to center of column.
  • Larger elution volumes and longer incubation times can sometimes increase yield.
  • For elution of DNA >10 kb, heat the DNA Elution Buffer to 50°C and extend incubation time to 5 minutes.
  • Multiple rounds of elution can also be performed.
Monarch PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) Reagents added incorrectly
  • Be sure that buffers have been reconstituted correctly, and that reagents have been added in the correct order.
Incomplete elution during preparation
  • Deliver Elution Buffer directly to center of column.
  • Larger elution volumes and longer incubation times can sometimes increase yield.
  • For elution of DNA >10 kb, heat the DNA Elution Buffer to 50°C and extend incubation time to 5 minutes.
  • Multiple rounds of elution can also be performed.
Low DNA quality

Monarch Plasmid Miniprep Kit (NEB #T1010)

Monarch Spin Plasmid Miniprep Kit (NEB #T1110)

 

 Plasmid degradation
  • Be cautious of strains with high levels of endogenous endonuclease (e.g., HB101 and JM 100 series).
Plasmid is denatured
  • Limit incubation with Plasmid Lysis Buffer (B2) to two minutes, as NaOH in the buffer can denature the plasmid.
Plasmid is contaminated with genomic DNA
  • Use careful inversion mixing after cell lysis to avoid shearing of host cell chromosomal DNA. Do not vortex.
  • Review our application note and protocol for removing residual gDNA in plasmid minipreps
RNA contamination
  • Incubate sample in neutralization buffer for the full 2 minutes. For cell culture volumes > 3 ml, increase the spin after neutralization to 5 minutes.
Improper storage
  • Elute DNA in DNA Elution Buffer or nuclease-free water, and store at -20°C. Do not store in solutions containing magnesium.
Low DNA performance

Monarch Plasmid Miniprep Kit (NEB #T1010)

Monarch Spin Plasmid Miniprep Kit (NEB #T1110)

 

 Ethanol has been carried over
  • Centrifuge final wash for 1 minute to ensure complete removal.
  • Ensure column tip does not come in contact with flow through.
Excessive salt in sample
  • Use both Plasmid Wash Buffers and do not skip wash steps.
Excessive carbohydrate has been carried over
  • Avoid strains with high amounts of endogenous carbohydrate (e.g., HB101 and JM 100 series). Be sure to follow protocol and include Plasmid Wash Buffer 1 step.
Monarch DNA Gel Extraction Kit (NEB #T1020) Gel slice not fully dissolved
  • Undissolved agarose may leach salts into the eluted DNA.
Ethanol has been carried over
  • Centrifuge final wash for 1 minute to ensure complete removal.
  • Ensure column tip does not come in contact with flow through.
Trace amounts of salts have been carried over
  • Ensure column tip does not come into contact with new tube for elution.
Monarch PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) Ethanol has been carried over
  • Centrifuge final wash for 1 minute to ensure complete removal.
  • Ensure column tip does not come in contact with flow through.
Trace amounts of salts have been carried over
  • Ensure column tip does not come into contact with new tube.