| PROBLEM |
PRODUCT |
CAUSE |
SOLUTION |
| No DNA purified |
Monarch Plasmid Miniprep Kit (NEB #T1010) |
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) |
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) |
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) |
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.
|
