Considerations for cDNA library construction
The construction of full-length cDNA library is divided into phage library and plasmid library, the two are similar. In any case, you should pay attention to the following aspects:
1. Ensure that a sufficient amount of high-quality starting RNA is obtained.
The amount of RNA required to construct a cDNA library is greater than that used for RACE and Northern blot. When the materials allow, the general kit recommends the use of purified total mRNA for reverse transcription, which is better than the direct reverse transcription of total RNA The constructed cDNA library is good, although the latter is not impossible. The old version of CLONTECH's SMART 4 ​​intermediate column requires that the total mRNA amount after purification should be about 0.05-0.5 micrograms, which requires a large amount of initial total RNA. Although some kits claim that as little as tens of nanograms of total RNA can also be used to construct a cDNA library, but this is for people with extremely scarce materials, but too little starting RNA will still more or less affect the success of library construction. Risk and representativeness of the library. As for the quality of RNA, if reverse transcription is used after purification of total mRNA, the requirements for impurities in total RNA are slightly loose, but the integrity of RNA is meticulous and requires no degradation. If the total RNA is directly used for reverse transcription, the quality of the total RNA is very high. Not only does the RNA need to be quite complete without degradation, but also the polyphenols, polysaccharides, proteins, salts, guanidine isothiocyanate and other impurities are less, the most Fortunately, it was extracted from the kit.
Second, the success of reverse transcription and the efficiency of reverse transcription are the keys.
This is the most expensive step in the construction of a cDNA library and a step in the qualitative change of nucleic acid. It turns easily degradable RNA into less easily degradable cDNA. The unsuccessful reverse transcription indicates the failure of a library scheme. The low efficiency of reverse transcription is that one part of the mRNA has been reverse transcribed, but a considerable part of the mRNA that should have been reverse transcribed has not been reversed; the second is that only a small part of the mRNA has been reverse transcribed. The structure is closest, and a large part of the mRNA is not fully reverse transcribed, and the total full-length cDNA is too small, which makes it difficult to construct a good full-length cDNA library. A small amount of mRNA degradation or incomplete reverse transcription has little effect on the titer of the library in the construction of kits such as SMART 4 ​​and manual methods, but it has a great impact on the full-length of the library. Invitrogen's new technology based on dephosphorylation, decapping, and reverse transcription after connecting RNA connectors (refer to its GeneRacer manual) is in principle the best method to ensure the final full-length cDNA is obtained, but it requires mRNA integrity Very high, in theory, it must be full-length mRNA with a cap structure and Poly A structure and complete reverse transcription to enter the library. It is very important to spot the cDNA concentration and molecular weight distribution after reverse transcription.
3. The operation of many steps after reverse transcription to packaging to the phage coat protein is relatively easy, but the chromatography column cDNA fractionation is critical.
A little care in this step will affect the success or the distribution characteristics of the obtained cDNA fragments. Be careful in this step, especially to ensure that the column can work properly by repeating suspension and test drip before adding cDNA. Focus on the addition and collection of cDNA. The amount of cDNA obtained at each level is very small, and the band pattern is very dark during the test. Therefore, use fresh transparent gel to detect. According to the test results, you must discard the cDNA that is too short (usually not under 400bp, because the short fragment Too much will seriously affect the subsequent connection conversion effect and library quality).
4. Both the phage library and the plasmid library require very high ligation efficiency of the vector and cDNA, and also require high efficiency of transfection or transformation of the ligation product into E. coli.
The efficiency of ligation is directly related to the success of the library construction. It is important to note that the ligation of the library is not the same as that of the general fragment cloning. The general fragment cloning connection is the connection of a fixed-length vector and a fixed-length target DNA, and the library connection is the connection of a fixed-length vector and a non-fixed-length target DNA. The target gene cDNA is more than 10kb long, and the short one is only 500bp or more short. As a result of a series of cDNAs of different lengths ligated together with the carrier, the ligation efficiency of cDNAs of different lengths is different. According to the experience of some experts, according to the classification results, cDNA groups of different lengths are consciously connected to vectors, and then transformed or transfected with E. coli, respectively, to complete the titer detection, and finally, libraries of different length levels are mixed together for Hybridization screening
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