Q List:
Q1: Why do glycerol preserved bacterial strains need to be activated by plate streaking? |
Q2: What culture medium should be used for bacterial cultivation? |
Q3: How to determine if the bacterial solution is normal after cultivation? |
Q4: What's the amount of bacterial solution corresponding to different culture media or culture media with different OD600? |
Q5: What is the reason of loose precipitation after the bacterial cells are lysed and centrifuged? |
Q6: What are the components of Elution Buffer? Can other reagents be used instead? |
Q7: What centrifuge does P1156 require for extraction? |
Q8: Can P1156 use filtration operation? |
Q9: Does Elution Buffer need to be preheated during elution? Does it need a second wash? |
Q10: Why does white precipitate appear in Buffer P2 reagent and how to handle it? |
Q11: How to determine the purity of the extracted plasmid? |
Q12: What is the reason for the low yield of P1156 after extraction? |
Q13: How to solve RNA contamination in extracted plasmid DNA? |
Q14: Why is P1156 easy to have RNA contamination? |
Q15: Why is the plasmid concentration extracted by P1156 high, but the electrophoresis band is weak? Why can't RNA contamination bands be seen during electrophoresis? |
Q16: Does RNA contamination affect cell transfection? |
Q17: What is the endotoxin level of plasmid extracted by P1156. Can it be directly used for cell transfection? |
Q18: How to further reduce the endotoxin of plasmids, improve the transfection efficiency of sensitive cells, or achieve injection grade? |
Q19: What is the principle of endotoxin removal? |
Q20: How to solve the problem of genomic DNA contamination in the extracted plasmid DNA? |
Q21: What are the advantages of P1156 compared to other competitors? |
Q1: Why do glycerol preserved bacterial strains need to be activated by plate streaking?
A: When bacterial strains are stored in glycerol solution, as time increases, some strains may lose copies. When directly inoculated from the preservation solution for amplification cultivation, these strains will become dominant bacteria, ultimately leading to a significant decrease in plasmid copy and the yield.
Q2: What culture medium should be used for bacterial cultivation?
A: It is recommended to use LB culture medium. YT or TB medium is a rich culture medium, and the rapid growth rate of bacteria is not conducive to sufficient plasmid copying, resulting in a decrease in plasmid production among the same number of bacteria. Therefore, it is not recommended to use YT or TB medium. If it is necessary to use YT or TB culture medium, adjust the amount of culture medium or alkaline lysis buffer appropriately after measuring OD600.
Q3: How to determine if the bacterial solution is normal after cultivation?
A: Measure OD600: LB culture medium, culture for 12-16 hours, OD600 should be 2.0-3.0.
Visual method: After 12-16 hours of cultivation, judge based on the turbidity of the bacterial solution or the weight of bacterial sediment after centrifugation. Do not use if the turbidity is too light or the amount of bacterial sediment is too small. Under the condition of : LB medium cultivate for 12-16 hours, OD600=3.0 and 150ml bacterial solution,after collected by centrifugation, the wet weight of the bacterial cells is about 0.6-0.8g.
Q4: What's the amount of bacterial solution corresponding to different culture media or culture media with different OD600?
A: Bacterial biomass=OD600 * volume of bacterial solution (ml). When the amount of bacterial solution of the high copy vector is 150ml, the bacterial biomass is 450
When using high-density culture medium (YT/TB), after measuring OD600, the corresponding bacterial solution dosage is 450/OD600. For example, after 16 hours of YT culture, if OD600=10, the operation flow of 45ml(450/10) culture medium is the same as that of 150ml LB culture medium
Q5: What is the reason of loose precipitation after the bacterial cells are lysed and centrifuged?
A: If the precipitation of bacterial cells after alkaline lysis and centrifugation appear loose or suspended, it's because of insufficient neutralization. The neutralizing solution doesn't fully penetrate into the interior of the precipitate. Due to the delayed neutralization, it is recommended to cultivate bacteria again before extraction to prevent genome contamination.
Refer to the table below and choose high-density or medium density lysis according to your own habits and requirements.
**Example: 150ml LB culture medium, OD600=3.0, bacterial biomass=450, wet weight of bacterial cells after centrifugation 0.6-0.7g.
Lysis Type |
Advantage |
Buffer P1 RNase A |
Buffer P2 |
Buffer LN3 |
||
Amount |
Phenomenon and mixing | Amount | Phenomenon and mixing | |||
High density |
Low supernatant volume, fewer passes through the column, saving time |
5ml | 5ml | The lysis solution is extremely viscous and the centrifuge tube needs to be repeatedly reversed up and down and rotated for fully lysis. Place for 3-5 minutes, during which repeatedly reverse, mix, and lightly shake, allowing the bacteria to fully lyse into a uniform, clumpless viscous liquid | 2.5ml |
The precipitate is large and compact, and needs to be inverted for 30-50 times quickly, accompanied by slight shaken to disperse the large precipitate into multiple small pieces, allowing the neutralizing solution to fully penetrate into the interior |
Medium density |
Easy to mix |
8ml | 8ml | The lysis solution is viscous.Repeatedly invert for 10-15 times. Place for 2-3 minutes, during which invert and mix several times. The total lysis time should not exceed 5 minutes | 4ml |
Easy to neutralize, invert and mix upside down until the large precipitate disperses into several small pieces |
Q6: What are the components of Elution Buffer? Can other reagents be used instead?
A: 10mM Tris, pH8.5 / Sterilized water / Buffer TE / enzyme free water can be used as substitutes of Elution Buffer. For sensitive applications, it is best to use freshly sterilized water. The production, packaging, and transportation of Elution Buffer cannot achieve complete sterility. P1156 uses PE bottles which cannot be directly sterilized.
Q7: What centrifuge does P1156 require for extraction?
A: Recommended use: Angle high-speed centrifuge (8000rpm). If high-speed centrifuge is not available, low-speed horizontal or angle centrifuge (3000~5000xg) is also acceptable.
For Low speed centrifuge (<4000xg): The column cannot be fully shaken to be dried and the elution buffer cannot be fully shaken off.
Dry the column: centrifuge at 3000-5000 x g for 15 minutes, remove the column and dry it at 55 degrees for another 10 minutes.
Elute: Add more elution buffer (>1ml) as there will be loss (0.3-0.4ml). The higher the speed, the less loss of elution buffer."
Q8: Can P1156 use filtration operation?
A: Using adapter Vac2, both column adsorping and washing steps can be replaced with filtration to reduce workload.
Q9: Does Elution Buffer need to be preheated during elution? Does it need a second wash?
For plasmid DNA<10KB, Elution Buffer does not need to be preheated. For large fragment plasmids>10KB, preheat Elution Buffer to 65°C
High speed centrifugation (>8000rpm): The elution buffer should not be less than 600μl, with a loss of ~0.15ml. It is recommended to transfer to the column for a second elution.
Low speed centrifugation (<4500kg):The elution buffer should not be less than 1ml, with a loss of ~0.3ml. It is recommended to transfer to the column for a second elution.
Q10: Why does white precipitate appear in Buffer P2 reagent and how to handle it?
A: Buffer P2 contains SDS, which crystallizes and precipitates at low temperatures. Before use, incubate at 37-55°C for 3-5 minutes to fully dissolve, then invert several times before use.
Q11: How to determine the purity of the extracted plasmid?
High quality plasmid DNA: OD260/280=1.80-1.96, OD260/230=1.8-2.5。
When OD260/280 exceeds 2.0, it indicates RNA contamination. Add RNase A to Buffer P1. RNA contamination can be removed by precipitation with isopropanol.
When dealing with low copy vectors or when the total amount of plasmids is less than 3μg, OD260/230 may be lower than 1.0. Trace amounts of guanidine salt pollution do not affect downstream applications and can be ignored.
Q12: What is the reason for the low yield of P1156 after extraction?
A: 1: Plasmid copy number: Different vectors can cause significant changes in yield due to copy number. The plasmid yield in 1ml LB bacterial solution is approximately 0.5-12μg (see the below table) >10KB vectors, cosmid, and lentivirus are generally low copy vectors. Before the experiment, the amount of bacterial cells can be controlled appropriately according to the type of carrier and culture medium to achieve the best effect.
Vector Structure | Vector Type | Origin of replication | Copy number | Classification |
1ml LB bacterial liquid yield |
pUC | Plasmid | pMB1 | 500-700 | High copy |
6μg |
pBluescript | Plasmid | ColE1 | 300-500 | High copy |
5μg |
pGEM | Plasmid | pMB1 | 300-400 | High copy |
4μg |
pTZ | Plasmid | pMB1 | >1000 | Ultra high copy |
10μg |
pBR322 and related | Plasmid | pMB1 | 15-20 | Low copy |
1.5μg |
pACYC and related | Plasmid | p15A | 13-20 | Low copy |
1.2μg |
pSC101 and related | Plasmid | pSC101 | ~5 | Ultra low copy |
0.5μg |
SuperCos | Cosmids | COLE1 | 13-20 | Low copy |
1.2μg |
pWE15 | Cosmids | COLE1 | 13-20 | Low copy |
1.2μg |
2: Bacterial strain: The glycerol preservation bacterial strain needs to be activated by streaking before cultivation. After culturing 2-3 tubes, observe whether the turbidity is normal, and select the highest turbidity for extraction.
3: Cultivation: the bacteria have not been fully expanded. After 14-16 hours of cultivation, the turbidity of the bacterial solution is insufficient, or the wet weight of the bacterial cells after centrifugation is not enough.
4: Insufficient resuspension after adding Buffer P1 resulted in a large number of bacteria not being fully lysed.
5: Buffer P2 may have precipitation. Heat to dissolve SDS, then invert and mix before use.
6: Insufficient lysis and neutralization. Adopting high-density lysis requires more mixing times and force. See Q5.
7: Insufficient elution: Add more elution volume and perform a second elution. Due to the loss of eluent, the larger the elution volume, the less loss.
Q13: How to solve RNA contamination in extracted plasmid DNA?
A: 1: Check if RNAse is added to Buffer P1. Buffer P1 containing RNAse must be stored at 2-8°C and can be stable for 6 months. After the expiration date, RNAse can be added and reused.
2: When using YT and TB culture media, conversion is required. See Q4.
3: RNA contamination can be removed by precipitation with isopropanol.
Q14: Why is P1156 easy to have RNA contamination?
A: Similar to most competitors, other kits which use ethanol or isopropanol as binding agents, also suffers from RNA contamination. When dealing with high copy vectors, RNA contamination is minimal and can be ignored. However, there may be significant RNA contamination when dealing with low copy vectors or some strains of bacteria. The nucleic acid concentration measured by the UV spectrophotometer is very high (virtual high), but the brightness of the electrophoresis band is weak, which do not match. When this situation occurs, precipitate again with isopropanol according to the additional procedure, or add more plasmids during transfection.
Q15: Why is the plasmid concentration extracted by P1156 high, but the electrophoresis band is weak? Why can't RNA contamination bands be seen during electrophoresis?
A: See Q14. Processing some strains and low copy vectors may result in RNA contamination in plasmids, causing high nucleic acid concentration when measured by UV spectrophotometry, but weak band brightness in electrophoresis.
The contaminated RNA fragments are very short (less than 100bp and single stranded) and do not bind to EB or dyes and run very fast, so RNA contamination bands are basically not visible after electrophoresis.
Q16: Does RNA contamination affect cell transfection?
A: RNA contamination does not affect transfection efficiency, but it can affect plasmid quantification, resulting in a lower actual amount of plasmid DNA used during transfection. More plasmids can be added for transfection based on the electrophoresis results.
Q17: What is the endotoxin level of plasmid extracted by P1156. Can it be directly used for cell transfection?
A: The plasmid DNA directly eluted with a column belongs to the transfection level and can be used for transfection in most cells. The endotoxin content is below 0.1Eu/μg.
Q18: How to further reduce the endotoxin of plasmids, improve the transfection efficiency of sensitive cells, or achieve injection grade?
A: The plasmid DNA directly extracted by P1156 belongs to transfection grade and is not recommended for animal injection.
If used for animal injection and high-sensitivity cell transfection, the additional process involves thoroughly removing endotoxins using Buffer ER2 extraction and dissolving plasmid DNA in freshly prepared sterilized ultrapure water.
Q19: What is the principle of endotoxin removal?
A: 1: Buffer ER2 contains Triton X-114, a classic endotoxin removal reagent that can bind with endotoxin molecules and efficiently remove endotoxins through aqueous two-phase extraction.
2: Wash Buffer contains unique endotoxin neutralizing solution, which can efficiently neutralize, wash and remove endotoxin molecules, achieving transfection level application.
Q20: How to solve the problem of genomic DNA contamination in the extracted plasmid DNA?
A: 1: The bacterial culture time is too long, and it is not recommended to exceed 16 hours.
2: The alkaline lysis operation is not standardized. After adding Buffer P2, the total lysis time should not exceed 5 minutes. When adding P2/LN3, do not vortex or vigorously shake, invert and rotate the centrifuge tube for mixing.
Q21: What are the advantages of P1156 compared to other competitors?
A: 1: Unique endotoxin removal washing buffer that efficiently removes endotoxins, reducing endotoxin levels by an order of magnitude and making it more friendly to cell transfection.
2: Using Trixon X114, remove endotoxins by dual mechanism , can meet the requirements of high-sensitivity transfection and injection.
3: Unique formula has less RNA contamination compared to competitors.
4: Provide a syringe type filter to completely filter and remove impurities after neutralization.