Plasmid extraction common problems and solutions
Answer plasmid extraction FAQ The coating stick is rubbed in alcohol and then burned. Can you guarantee that the bacteria used will be burned? Reference opinion: The coating stick can be preserved in alcohol, but alcohol cannot be sterilized immediately. After simmering the alcohol, burn it for a little while, burning alcohol instead of coating the stick. It is recommended to apply the stick or dry it for a long time, then cool it and recoat it. When multiple transformations are performed at the same time, several coating bars are applied to avoid cross-contamination. The bacteria that were not problematic were originally sequenced, and the plasmid size or sequence was abnormal after shaking at 37 °C. References: This situation is less likely to occur, often in larger plasmids or in more specific sequences. Solution: 1. Lower the culture temperature, culture at 20-25 °C, or culture at room temperature can significantly reduce the probability of occurrence. 2. Use some special strains, such as Sure strain, which lacks some recombinases, such as rec, to make plasmid replication more stable. 3, plasmid extraction has a lack of enzyme digestion is caused by the high concentration of NaOH in solution II, please pay attention! [There are two ways to determine if the growth of the bacteria is normal before the plasmid is raised: 1. Use your sense of smell. As long as you do the experiment, you can smell the smell of E. coli. The fresh E. coli is slightly pungent, but it is not disgusting. In the muddy liquid, you only need to feel the smell is very incomparable or smellless when you get together, you can compare with the normal bacteria. 2. Visually observe the activated strain. For the abnormal growth of the bacterial liquid, the plate was verified or diluted to a concentration low enough to coat the plate. The next day, the growth of the monoclonal medium was observed. The normal form of DH5A grown on the LB plate was about 1 mm at 37 ° C for 16 hours, and the color was white. Translucent, moist round plaque, if it is observed that the growth is too fast, the color is yellowish, it is basically abnormal. 】 No plasmid or plasmid yield is low, how to solve it? References: 1. E. coli aging: After plating the plate, the new colonies are re-selected for liquid culture. 2. Low copy number of plasmid: Since the amount of plasmid DNA extracted by using a low copy number vector is low, a high copy number vector having the same function can be replaced. 3. No plasmid in the bacteria: Some plasmids themselves cannot be stably present in some strains, and may cause plasmid loss after repeated transfer. For example, cosmids are unstable in Escherichia coli for long-term storage, so do not switch frequently, and single colonies should be inoculated each time. In addition, check whether the concentration of antibiotics used for screening is correct. 4, alkali lysis is not sufficient: the use of too much bacterial culture solution, will lead to insufficient cell lysis, can reduce the amount of bacteria or increase the amount of solution. For low copy number plasmids, increasing the amount of cells and doubling the solution during extraction can help increase the amount of plasmid extracted and improve the quality of the plasmid. 5, improper use of the solution: Solutions 2 and 3 may appear turbid at low temperatures, should be placed at 37 ° C for a while until dissolved into a clear solution, can be used. 6. Adsorption column overload: The adsorption capacity of the adsorption column is different in different products. If the amount of plasmid to be extracted is large, please extract it several times. If an enrichment medium, such as TB or 2×YT, is used, the volume of the bacterial solution must be reduced; if the plasmid has a very high copy number or the host strain has a high growth rate, the volume of the LB medium must be reduced. 7. The plasmid is not completely dissolved (especially when the plasmid is large): When eluting the lysed plasmid, the temperature may be appropriately increased or the dissolution time may be prolonged. 8. Ethanol residue: After washing the rinse solution, the residual liquid should be removed by centrifugation as much as possible, and then the elution buffer should be added. 9. The eluent is not added correctly. The eluent should be added to the center of the silica gel membrane to ensure that the eluent will completely cover the surface of the silica membrane to achieve maximum elution efficiency. 10. Eluent is not suitable: DNA can only be eluted in a low salt solution, such as elution buffer EB (10 mM Tris-HCl, 1 mM EDTA, pH 8.5) or water. The elution efficiency is also dependent on the pH and the maximum elution efficiency is between pH 7.0 and 8.5. When eluting with water, ensure that its pH is within this range. If the pH is too low, the elution amount may be low. When the sterilized distilled water or the elution buffer is heated to 60 ° C during elution, it is advantageous to improve the elution efficiency. 11. The elution volume is too small: the elution volume has a certain influence on the recovery rate. As the elution volume increases, the recovery rate increases, but the product concentration decreases. The elution volume can be increased in order to obtain a higher recovery rate. 12. The elution time is too short: the elution time will also have a certain impact on the recovery rate. A good result can be achieved by placing it for 1 min at the time of elution. After the bacteria were centrifuged and added to the solution I, the cells were found to be flocculated unevenly or in a fine sand shape. References: 1. It is likely that the bacteria are lysed, the culture time can be reduced or the plate culture can be tried. The colonies are washed with PBS before the plasmid extraction, and the bacteria growth on the solid medium is better. 2. The plasmid extraction process is largely determined by the growth of the bacteria. The newly activated bacteria are better than the bacteria cultured at minus 80 °C, and the long-preserved strain may cause low plasmid concentration. Unknown reasons such as loss. 3. Judging whether the growing bacterial liquid is normal, it can be observed with the naked eye, and the fresh culture liquid is shaken in the bright light. If the bacterial liquid is found to be floated, the condition is very good. If it is found to be muddy, it will not see flocculation, but it will feel very turbid, and may not give a good plasmid, or no plasmid. 4, the bacterial liquid should not grow too thick, the speed of the shaker should not be too high. It can be achieved by reaching OD600 1.5 (especially for the extraction of the kit). If it is only a simple enzyme digestion, no phenol chloroform extraction is required at all (safety consideration, prudent), as long as the ratio of solution I/II/III is appropriate, the tube The process carefully draws up without too many magazines. Why did the bacteria gradually become clarified by turbidity after adding solution II? The proposed band is almost absent, but the RNA is very bright (no RNase added)? References: Solution II is mainly NaOH, if the bacterial solution is not clarified by turbidity. 1. It may be because the solution is not stored properly, or the solution bottle cap is not covered in time, causing it to absorb CO2 in the air. RNA is more abundant in the cells, and a relatively small amount of cells are cleaved, which may have a clearer band than DNA. 2, may be a large amount of bacteria, after adding solution II, the bacteria can not be completely lysed, so there is no clear, which will lead to low yield of the plasmid. 3. It may be that the copy number of the plasmid is not high and the plasmid yield is not high. If you are using your own reagents, it is recommended to do the middle or big mention; or buy a kit. Use your own reagents, no RNase, and finally the RNA is very bright. To remove it, use a better RNase. 4. If it is not the cause of the reagent, it may be that the membrane protein changes (the quantity becomes more) during the expression of the plasmid. It is difficult to use the alkaline lysis method, and other relatively vigorous methods (such as high temperature or low temperature grinding) can be tried, and then Use general distribution. 5. The plasmid may be discarded with ethanol. After adding solution II, the bacterial liquid is still cloudy, or the turbidity is not significantly changed. The cracking is not complete, refer to the insights: 1. The problem may be that it occurs on solution II. First look at whether 10% SDS is clarified? Is NaOH effective? If you are using a kit, first check if Solution II is clear and there is no precipitation. 2. It may be that the concentration of the bacteria is very high, and the volume of the solution I/II/III is appropriately adjusted. 3, may be "microbial" pollution, if the bacterial growth is abnormally fast, it may be contaminated by bacteria. This situation generally shows the same resistance to the target bacteria, the growth rate is abnormal, the plasmid can be proposed, and the band of the gel is also abnormally bright, but the product is not the plasmid that one wants, and special attention should be paid. Wheeled Hose Reel Cart,Hose Cart With Wheels,Large Hose Reel Cart,Garden Hose Cart With Wheels NINGBO QIKAI ENVIRONMENTAL TECHNOLOGY CO.,LTD , https://www.hosereelqikai.com