cDNA library construction
cDNA library construction
The cDNA library construction can be: (1) used to isolate full-length genes for gene function studies; (2) screen target genes and use them directly for expression; (3) provide probes for constructing molecular markers for interlocking mapping.
Operation method
cDNA library construction
Principle
A cDNA library is a collection of clones formed by ligating the cDNA fragments formed by reverse transcription of all mRNA transcribed during a certain developmental period of an organism to a certain vector. The basic principle of classical cDNA library construction is to use Oligo(dT) as a reverse transcription primer, or use random primers to add appropriate junctions to the synthesized cDNA, and then ligated into the appropriate vector to obtain the library. The basic steps include: (1) mRNA purification: obtaining high-quality mRNA is one of the key steps in constructing a high-quality cDNA library. (2) Synthesis of the first strand of cDNA. (3) Synthesis of the second strand of cDNA. (4) Modification of double-stranded cDNA. (5) Molecular cloning of double-stranded cDNA. (6) Amplification of cDNA libraries. (7) Identification and evaluation of cDNA libraries.
Materials and Instruments
mRNA Move I. Superscipt II-RT synthesis of first strand 1. Add x ul mRNA (about 500 ng), 1 ul Xho I Primer (1.4 ug/ul) into an RNase-free 0.2 ml PCR tube. (5’ GAGAGAGAGAGAGAGAGAGAACTAGTCTCGAGTTTTTTTTTTTTTTTTTT…3’)、11-x ul RNase-free water (>500 ng mRNA is divided into n-tubes (500 ng/tube) for first strand synthesis, and after first strand synthesis is complete, the n-tubes are combined into a single tube for second strand synthesis). The first strand will be synthesized in n-tube (500 ng/tube). 2. After mixing, react at 70°C for 10 minutes. 3. 3. Immediately after completion of the reaction, place the reaction system on ice for 5 min. 4. 4. Centrifuge the system briefly and add the following reagents sequentially: (1) 4 ul 5×first strand buffer (2) 2 ul 0.1 M DTT (3) 1 ul 10 mM dNTP (prepared by yourself). 5. 5. After mixing and slightly centrifuging the reactants, leave at 42°C for 2 minutes. 6. 6. When the reaction is completed, add 1 ul Superscipt II-RT while it is still hot and mix well. 7. 7. React at 42℃ for 50 minutes, then inactivate the reverse transcriptase at 70℃ for 15 minutes. II. Synthesis of the second strand of cDNA 1. After the completion of the first-strand reaction, take 2ul of the first-strand product and store it in the refrigerator at -20℃ for electrophoretic detection. The remaining products were combined, mixed well, and then the following reagents (promega) were added sequentially: (1) 20 ul 10×DNA Polymerase I buffer (2) 6 ul 10 mM dNTP (own preparation) (3) x ul dd H2O (4) 1 ul RNase H (2 U/ul) (5) 10 ul DNA Polymerase I (10 U/ul) The total system is 200 ul. 2. After mixing, react at 16°C for 2.5 hours. 3. Inactivate at 70°C for 10 minutes. 4. After completion of the reaction, 200 ul cDNA second strand reaction system was obtained and this system was placed on ice. 5. Take 2 ul of the second-strand product and identify it by electrophoresis together with the preserved first-strand product. At the same time on the 1kb ladder to determine the size range of the double strand. Note: The electropherograms of one-stranded and two-stranded cDNAs are smear, and the two-stranded cDNAs are slightly larger than the one-stranded cDNAs. III. Double-stranded cDNA end leveling 1. In the second strand reaction system, add the following reagents (promega) in sequence: (1) 6 ul 10 mM dNTP (2) 2 ul T4 DNA Polymerase (8.7 U/ul) (3) 2 ul BSA (10 mg/ml) 2. Slightly centrifuge and mix the reactants, react at 37°C for at least 30 minutes, then inactivate at 75°C for 10 minutes. 3. Add an equal volume of phenol/chloroform/isoamyl alcohol and centrifuge at 13,000 g for 5 min at room temperature after vigorous shaking. 4. After centrifugation, aspirate the supernatant into another 1.5 ml eppendorf tube, add an equal volume of chloroform, mix several times upside down and centrifuge at 13,000 g for 5 minutes at room temperature. 5. Aspirate the supernatant into another eppendof tube, add 1/10V 3M NaAc (PH5.2) and 2.5 V pre-cooled anhydrous ethanol, mix well, and leave it at -20℃ overnight to precipitate double-stranded cDNA. 6. On the second day, yesterday's precipitate was centrifuged at 13 000 g for 60 min at 4°C to fully precipitate double-stranded cDNA. 7. After centrifugation, the supernatant was discarded, the precipitate was washed with 1 ml of 70% ethanol, and centrifuged at 13,000 g for 5 min at room temperature. 8. After centrifugation, discard the supernatant and dry the precipitate until it has no ethanol odor. Note: Steps 3 and 4 can be replaced by PCR purification kits. PCR purification kit operation procedures 1. Before using the solution PE should be added to the appropriate volume of 95% -100% ethanol, mix well. 2. 2. Add 5 times the volume of buffer PB to 200 ul of two-stranded complementary products, mix well. 3. 3. Add to spin column and centrifuge at 13 000 rpm for 1 min. 4. 4. Add 0.75 ml buffer PE and centrifuge at 13 000 rpm for 1 min. 5. 5. Centrifuge again at 13,000 rpm for 1 min. 6. 6. Place the spin column in a new centrifuge tube, add 50 ul buffer EB, and allow to stand for 10 min. 7. Centrifuge at 13 000 rpm for 2 min. 8. 8. Add 30 ul buffer EB and let stand for 10 min. 9. 9. Centrifuge at 13 000 rpm for 2 min. 10. 10. Add 1/10 volume of 3M NaAc, 2.5 times volume of anhydrous ethanol, mix well, and precipitate at -20°C overnight. V. EcoR I adaptor splicing 1. Add 9 ul of EcoR I adaptor (400 ng/ul) to the double-stranded cDNA precipitate and leave it at 4℃ for at least 30 minutes to fully dissolve the cDNA precipitate. 2. After dissolution is complete, add the following reagents sequentially: (1) 1.2 ul 10×Ligase Buffer (2) 1 ul 10 mM rATP (3) 1 ul T4 DNA Ligase (4 U/ul). 3. 3. After mixing, ligate at 4°C for 3days or at 8°C overnight. Phosphorylation of double-stranded cDNA ends and Xho I digestion. 1. After the ligation reaction is completed, leave the reaction system at 70℃ for 15 minutes to inactivate the T4 DNA Ligase. 2. 2. Slightly centrifuge the reaction material to the bottom of the tube and leave it at room temperature for 5 minutes, then add the following reagents: (1) 1 ul 10×Ligase Buffer (2) 1 ul 10 mM rATP (3) 6 ul dd H2O (4) 1 ul T4 PNK (10 U/ul) 3. React for 30 min at 37°C, then inactivate for 15 min at 70°C. 4. Centrifuge the reaction slightly. 4. centrifuge slightly to concentrate the reaction to the bottom of the tube. 5. Leave at room temperature for 5 minutes; then add the following reagents: (1) 4 ul Xho 10×Buffer (2) 2 ul BSA (3) 5 ul ddH2O (4) 8 ul Xho I (10 U/ul) 6. The reaction was carried out at 37 °C for 1.5 h, followed by inactivation of the enzyme at 65 °C for 10 min. 7. The reaction was completed and double-stranded cDNA synthesis was finished. Place at 4°C and prepare for recovery. Gel recovery of cDNA 1. Prepare a small gel plate (one plate for each sample): 1% agarose gel, 2 ul EB/300 ml gel. 2. 2. Take the samples stored at 4℃, 40 ul/well. 3. 3. Electrophoresis at 50 V for 1 h. 4. 4. 500-1 kb, 1.0-2.0 kb and 2.0-4.0 kb cDNA fragments were cut under UV light. Put them into labeled 1.5 ml centrifuge tubes. 5. 5. Weigh the gel and add three times the volume of buffer QXI (e.g., 300 ul buffer QXI to 100 mg gel). 6. Take a water bath at 50°C for a few minutes until the gel is completely melted. Resuspend QIAEX II by flicking it with your finger and add 5 ul of QIAEXII to each tube. 7. 50°C water bath for 10 min, remove every 2 min and mix upside down several times to keep QIAEX II in suspension. 8. 4°C, 13 000 rpm. 8. 4°C, 13 000 rpm for 30 s (discard supernatant, shake in centrifuge and aspirate supernatant). 9. Add 500ul of buffer QXI and flick the bottom of the tube to resuspend QIAEX II. 10. 10. Centrifuge and remove supernatant (as in procedure 8). 11. 11. Add 500 ul buffer PE, resuspend QIAEX II, centrifuge for 30 s, and remove supernatant. 12. 12. Add another 500 ul buffer PE, resuspend QIAEX II, centrifuge for 30 s, discard supernatant, shake in centrifuge, and aspirate supernatant. 13. Blow-dry on ultra-clean bench (until no ethanol odor), add 10 ul elution buffer, resuspend QIAEX II, let stand for 5 min, 13 000 rpm, 30 s. Aspirate supernatant and place on ice. 14. 1 ul of supernatant was taken for electrophoresis, and molecular weight standards (1 kb ladder) and DNA content standards (10 ng, 20 ng) were made as controls. 15. Store the recovered cDNA at -20℃, and according to the electrophoresis result, take an appropriate amount of DNA for ligation. Caveat 1. RNA extraction (e.g., guanidine isothiocyanate, guanidine hydrochloride-organic solvent method, hot phenol method, etc., the choice of extraction method depends mainly on different samples). 2. To construct a high-quality cDNA library, it is essential to obtain high-quality mRNA, so mRNA samples must be handled carefully. 3. since RNAase is present in all organisms and is resistant to physical environments such as boiling, it is important to establish an RNAase-free environment for the preparation of good quality RNA. 4. the choice of a suitable enzyme for library construction is important. 4. For library construction, it is important to choose a suitable vector.λ-phage, this is becauseThis is because λ-This is because λ-phage DNA has a 12-nucleotide sticky end, which can be used to construct the Coase plasmid, which can hold a large portion of exogenous DNA. 5. Soak the electrophoresis tanks, plates and combs in 1% HCl overnight before gel recovery. 6. Stabilize the voltage during gel recovery. Common Problems I. Key factors for cDNA construction succes 1. Ensure that a sufficient amount of high-quality starting RNA is obtained For more product details, please visit Aladdin Scientific website.
DTT Distilled Water dNTP EDTA SDS Ethanol Polymerase Agarose DNA Polymerase BSA T4 DNA Ligase Phenol Chloroform Isoamyl Alcohol PCR Purification Kit
Ice Maker Centrifuge Water Bath Electrophoresis Instrument Centrifuge Tubes Pipette Gun Incubator
Older versions of CLONTECH's SMART 4 have intermediate columns that require a purified total mRNA amount of preferably around 0.05-0.5 µg, which requires a higher amount of starting total RNA. Although some kits claim that cDNA libraries can be constructed with as little as a few tens of nanograms of total RNA, this is for those who have extremely scarce materials, but too little starting RNA will still affect the risk of successful library construction and the representativeness of the library to a greater or lesser extent.
As for the quality of the RNA, if purified total mRNA is used for reverse transcription, the requirements are slightly less stringent in terms of impurities in the total RNA, but meticulous in terms of RNA integrity, which is required to be undegraded. If total RNA is directly used for reverse transcription, the quality of total RNA is very demanding, requiring not only that the RNA be quite complete and undegraded, but also that there be few impurities such as polyphenols, polysaccharides, proteins, salts, guanidine isothiocyanate, etc., and preferably that it be extracted by the kit.
Reverse transcription efficiency is not high in one is part of the mRNA was reverse transcribed, but there is still a considerable part of the mRNA should be reverse transcription of the mRNA has not been reversed; Secondly, only a small part of the mRNA was reverse transcribed through the nearest to the hat structure, and a large part of the mRNA is not reverse transcribed completely, the total full-length cDNA is too little, which is difficult to build a good full-length cDNA library.
A small degree of mRNA degradation or incomplete reverse transcription has little effect on the titer of the library in kits such as SMART 4 and manual construction, but it has a great impact on the full-length of the library. Invitrogen's new technology based on dephosphorylating, de-capping, and reversing the transcription after RNA junction junction ligation (refer to its GeneRacer instruction manual) is the best way to ensure that the final full-length cDNA can be obtained in principle. In principle, it is the best way to obtain full-length cDNA, but the requirement for the integrity of mRNA is very high. Theoretically speaking, it must be full-length mRNA with cap structure and Poly A structure, and reverse transcription must be complete before it can enter into the library. It is important to check the concentration and molecular weight distribution of the cDNA after the completion of reverse transcription.
The amount of cDNA obtained for each level is very small, and the band pattern is very dark when detected, so it should be detected with freshly made transparent thin gel, and cDNAs that are too short must be discarded according to the detection results (usually not below 400bp, because too many short fragments will seriously affect the ligation transformation effect and library quality later).
General fragment cloning ligation is a fixed-length vector ligated to a fixed-length target DNA, while library ligation is a fixed-length vector ligated to a non-fixed-length target DNA, and the target gene cDNA is more than 10kb long, while the short one is only 500bp or shorter.
As a result of a series of cDNAs of varying lengths being ligated together with vectors, the efficiency of ligation varies for different lengths of cDNAs. The experience of some experts is to consciously ligate groups of cDNAs of different lengths with vectors separately according to the grading results, then transform or transfect E. coli separately, complete the titer assay separately, and finally mix libraries of different length classes together for hybridization screening.