This protocol describes the method of exon amplification using the mammalian shuttle vector pSPL3 as an example, which is divided into the following five stages. Stage 1: Library construction; Stage 2: Transfection of library into COS-7 cells by electroporation; Stage 3: mRNA extraction; Stage 4: Reverse transcription PCR; Stage 5: Cloning analysis. This experiment was obtained from Molecular Cloning Laboratory Guide (3rd edition), author: Peitang Huang.
Operation method
Exon capture and amplification
Materials and Instruments
E. coli strain HB101 Plasmid pSPL3 COS-7 Cell Vector pBluescript II E. coli DH5α Move Stage 1: Library Construction For more product details, please visit Aladdin Scientific website.
pSPL3 polyclonal site mapping Restriction endonuclease PvuII T4DNA ligase LB agar plate Sticky vector TE SOC medium Agarose gel LB broth medium PBS Trypsin-EDTA solution DNA DMEM DEPC Treated water Ethanol NaCl TMK buffer SDS DTT dNTP solution Bst XI EcoR V Mo-MLV Anti Transcriptase Taq DNA polymerase DNA uracil glycosylase RNase inhibitor
Water baths Centrifugal heads Electroporation transfection equipment to Electrotransfection baths Tissue culture dishes Wide-mouth spacer heads Cell scrapers or rubber scrapers Polystyrene centrifuge tubes Thermal cyclers Multi-channel spacers or spiking devices
Materials
Buffers and solutions
Dilute the storage solution in the appropriate proportion.
ATP (10 mmol/L)
Phenol: chloroform (1:1,V/V)
TE(pH8.0)
Enzyme & Buffer
Pvu II
Restriction endonuclease
The pSPL3 polyclonal site map is shown in Figures 11-16.
T4DNA ligase
Gel
Agarose gel (0.9% m/V), prepared with TAE
See step 3
Agarose gel, prepared
see step 1
Nucleic acids and oligonucleotides
Mucoid vectors, E. coli artificial chromosome or yeast artificial chromosome DNA, containing regions of target genomic DNA.
Recombinant DNA purification methods for analysis are described in Chapter 4.
or genomic DNA
Prepare by one of the methods provided in Chapter 6.
Medium
LB agar plates containing 50ug/ml ampicillin
See Step 8 to determine the appropriate size of plate.
LB Broth Medium with 50ug/ml Aminobenzylpenicillin
SOC medium
Specialized equipment
Water baths with preset temperatures of 15°C and 65°C
Additional reagents
The reagents required in step 8 of this program are listed in Chapter 1, Option 20.
The reagents required for transformation in step 6 of this protocol are listed in Chapter 1, Scheme 23, 24, or 25.
Reagents required for steps 10 and 12 of this protocol are listed in Chapter 1, Scheme 1 or 9.
Vectors and Strains
E. coli strain HB101, in the transformed susceptible state
Prepared according to Chapter 1, protocol 23, 24 or 25.
Plasmid pSPL3
Life Technologies provides plasmid pSPL3 either alone or as a kit component.
Methods
1. pSPL3 was subjected to restriction enzyme digestion for insertion into genomic DNA (see Figures 11-16 for pSPL3 polyclonal site mapping). The linearized plasmid DNA was dephosphorylated, electrophoresed and the purified plasmid recovered from the gel.
It is important that the plasmid DNA is digested completely and that the product is a single fragment of approximately 6 kb in size.
2. Digest 1-2ug of genomic DNA or recombinant plasmid containing the target genomic DNA with the same restriction enzyme as in the previous step.
3. Analyze 10% of the genomic digest by 0.9% agarose gel electrophoresis (TAE system).
The genomic DNA must be completely cleaved without degradation. When analyzing by gel electrophoresis, watch for contaminating bacterial (or yeast) genomic DNA, which usually appears as a faint background band.
4. The genomic DNA digestion system is terminated at 65°C for 15 min, and the DNA is recovered by phenol/chloroform extraction and standard ethanol precipitation. the product is dissolved in TE (pH 8.0) at a concentration of 100ug/ml.
5. Ligate the genomic DNA into the vector by mixing the following components:
Enzymatically digested genomic DNA 150ng
150ng digested and dephosphorylated PSPL3 50ng
10x Ligation Buffer 1ul
T4 Phage DNA Ligase 10 Weiss Units
Add H2O to 10ul
Incubate at room temperature for 2~3 h or overnight at 15°C.
If ATP has been added to the 10X Ligation Buffer component, a larger volume of vector or exogenous DNA can be added to the reaction mixture, or if a commercial ATP-containing ligation buffer is used, ATP does not need to be added. make sure to introduce a control with only vector DNA, which is important for evaluating the quality of the constructed libraries.
6. Transform 40ul of HB101 receptor cells with the ligation reaction product.
Ensure that a positive control (vector DNA) and a negative control (no DNA system) are transformed at the same time to evaluate the transformation efficiency.
Important: Due to the instability of pSPL3 replication in other strains, the use of HB101 as a host for library and vector amplification is mandatory (Churchand Buckler1999).
7. Add 800ul of SOC medium to the post-transformation broth and incubate at 37°C for 30-45 min to allow expression of the plasmid-encoded antibiotic resistance marker gene.
8. Shake the bacteria overnight at 37°C in the presence of ampicillin to amplify the library.
If using a single plasmid: apply 100ul of the transformation mixture to an LB agar plate containing 50ug/ml ampicillin. Add 2 ml of LB broth liquid medium containing 50ug/ml ampicillin to the remainder to continue incubation.
The transformation efficiency can be evaluated by incubating a small amount of the transformants on LB plates.
If using BAC, PAC or mucoid library: spread all transformation mixture on multiple 50ug/ml ampicillin LB agar plates (150 mm).
This treatment is to eliminate dominant growth of certain clones due to nutrient competition and to make the constructed library more representative.
9. Estimate ligation efficiency by comparing the number of recombinants to the number of nonrecombinants (i.e., compare to the number of clones obtained by transforming HB101 with a ligation mixture of only pSPL3).
Recombinants should be at least 500-fold to 1,000-fold more numerous than non-recombinants.
10. If library amplification is performed on 150 mm plates, proceed directly to step 11. For genomic libraries amplified in liquid broth medium, purify the plasmid using a standard alkaline denaturation method (see Chapter 1, Scheme 1) or a plasmid extraction kit and proceed to step 13.
11. If the entire library is spread on a large LB agar plate, add 10 ml of broth medium to submerge the plate and carefully scrape the colonies from the surface of the agar, minimizing the amount of agar scraped into the broth.
12. Purify the plasmids from the samples obtained in step 11 using the modified standard alkaline denaturation small volume plasmid extraction procedure, and sequentially add the reagents to the bacterial precipitate by adjusting the amount of alkaline lysate I (300ul), lysate II (600ul) and lysate III (450ul). To simplify the procedure, after the addition of Base Lysate I, the bacterial suspension was pipetted into a 2 ml centrifuge tube so that the rest of the procedure could be done in this tube. After addition of Lysis Solution III and centrifugation, the crude plasmid supernatant was bisected into two 1.5 ml centrifuge tubes. The supernatant is extracted with phenol: chloroform and chloroform, the resulting supernatant is precipitated with isoamyl alcohol, and the DNA resuspensions from the two tubes are combined. This step can also be done with commercial plasmid extraction reagents (Chapter 1, Scheme 9).
13. Take part of the purified plasmid DNA and digest it with Pvu II, and observe the quality of the constructed library by electrophoresis.
The Pvu II cleavage site is close to the polyclonal site of the plasmid. The electrophoresis of empty vector digests showed 4kb and 2kb bands. Child clones should still show a clear 4-kb band after cleavage, but the 2-kb band should be faint or absent. a clear 2-kb band suggests that the library is of poor quality or, less likely, that the genomic DNA has been cleaved by Pvu II into mostly 2-kb fragments (Fig. 11-17). If the quality of the library is satisfactory, it can be used in the next step of the experiment. 
Stage 2: Transfection of COS-7 Cells with the Library by Electroporation
MATERIALS
Buffers and Solutions
Dilute the storage solution in the appropriate proportion.
Divalent cation-free phosphate buffer solution (PBS)
Enzymes and buffers
Trypsin-EDTA solution
Nucleic Acids & Oligonucleotides
DNA and Oligonucleotides
Plasmid DNA preparation for transfection is described in Stage 1, Steps 10-12 of this protocol.
Plasmid vector for control (step 3)
Medium
Dulbecco's modified Eagle's medium with 10% heat-inactivated fetal bovine serum (DMEM)
Centrifugal rotors
Sorvall H1000B head or equivalent
Specialized equipment
Electroporation transfection equipment and 0.4 cm wide electrotransfection tanks
Tissue culture dish (100 mm)
Wide mouth sampler head
Cells and Tissues
COS-7 Cells
Cells must be cultured in DMEM containing 10% heat-inactivated fetal bovine serum until 75%-85% dense. See Spector et al. (1998b) for details of cell handling, including tryptic digestion.
Methods
1. Wash monolayers of C0S-7 cells with divalent cation-free PBS. Cells were digested off the surface of the dish by trypsin-EDTA treatment. cells were recovered by centrifugation at 4°C for 10 min at 250 g (equivalent to 1100 r/min on a Sorvall H1000B turntable).
Important: COS-7 cells must be kept pre-cooled throughout the process.
See Spector et al. (1998b) for details on tryptic digestion of cells.
2. Dissolve the DNA sample for transfection in divalent cation-free PBS and adjust to 100 ul.
The amount of DNA required for transfection should be optimized according to Chapter 16, Protocol 5.
3. Resuspend 4X106COS-7 cells with 700ul of divalent-free PBS. Mix cells and DNA in a pre-cooled electrotransfection tank (0.4 cm wide in the tank). Place the mixture on ice for 10 min.
The presence of divalent cations in the PBS system will result in arcing during electrotransfection, which can disrupt electroporation only.
Ensure that empty vector DNA is applied for electrotransformation as an experimental control.
4. Carefully resuspend cells and perform electroporation transfection experiments at 1.2kV (3kV/cm) and 25uF. Quickly reposition the electrotransfection bath on ice and leave for 10 min.
Electroporation conditions are optimized as described in Chapter 16, Protocol 5.
5. 1x106 of electroporated snarls are transferred with a wide-mouth spiker tip into 100 mm tissue culture dishes, each of which has been filled with 10 ml of DMEM medium containing 10% heat-inactivated fetal bovine serum preheated to 37°C. The sample is then transferred to a 100 mm tissue culture dish. Incubate in an incubator at 37°C, 5%~7% CO2 and certain humidity for 48~72 h.
Stage 3: mRNA extraction
Material
Buffer and solution
Dilute the storage solution in the appropriate proportion.
DEPC Treated Water
Ethanol
NaCl (5mol/L)
Phenol
Phenol: chloroform
Divalent cation-free phosphate buffer solution (PBS)
SDS (5% m/V)
TMK buffer
10 mmol/L Tris-HCl (pH 7.5)
10 mmol/LKCl
1 mmol/LMgCl2
Triton X-100 (10% V/V) or Nonidet P-40 (4%, V/V)
Centrifugal rotor head
Sorvall H1000B turntable or equivalent
Specialized equipment
Cell scraper or rubber scraper
Polystyrene centrifuge tube (15 ml), pre-cooled on ice to 0°C
Cells and Tissues
COS-7 cells transfected with recombinant pSPL3 and non-recombinant pSPL3 respectively
Methods
1. Wash transfected COS-7 cells in dishes three times with ice-cooled calcium- and magnesium-free PBS. Keep the dishes on ice during washing.
2. Add 10 ml of pre-cooled PBS to each dish, keep the dish on ice and carefully scrape off the cells.
3. Transfer the cell suspension to a pre-cooled 15 ml polystyrene centrifuge tube.
The use of clear polystyrene centrifuge tubes facilitates the recovery of cytoplasm without nuclei in Steps 8 and 9.
4. Recover the cells by centrifugation at 300 g (equivalent to 1200 r/min on a Sorvall H1000B turntable) at 4°C for 8 min.
5. Pour off as much supernatant as possible and remove any remaining supernatant with a spiker.
6. Resuspend 1X106 to 2x106 COS cells in 300ul of TFK buffer and place on ice for 5 min.
7. Add 15ul of 10% Triton X-100 or 4% Nonidet P-40 and place on ice for 5 min.
Addition of a nonionic detergent may result in a slight cracking of the plasma membrane while the nuclear membrane remains intact.
8. Centrifuge at 500 g (equivalent to 1500 r/min on a Sorvall H1000B turntable) for 5 min at 4°C to precipitate the nuclei.
9. Transfer the supernatant to a pre-cooled 1.5 ml mini-centrifuge tube.
IMPORTANT: Use extreme caution when removing the supernatant and do not touch the nuclei. Rupture of the nuclear membrane will result in genomic DNA contamination and the sample will be very sticky and difficult to aspirate.
10. Add 20ul of 5% SDS and 300ul of phenol, shake vigorously and centrifuge at maximum speed for 5 minutes to separate the sample into organic and aqueous phases.
11. Transfer the aqueous phase into a small 1.5 ml centrifuge tube containing 300ul of phenol: chloroform and centrifuge vigorously for 3 min at room temperature on maximum speed.
12. Pipette the upper aqueous phase into a pre-cooled 1.5 ml mini-centrifuge tube and add 120 of 5mol/L NaCl and 750ul of ethanol. Place at -20°C for 2-3 h or -80°C for 30 min to precipitate RNA.
Nucleic acids are more stable in ethanol than in water. For long-term storage, RNA should be frozen in ethanol at -80°C (see the column at the end of Scheme 1, Chapter 7, "Preservation of RNA").
Recover the RNA by centrifugation at 13.4°C for 10 min at maximum speed.
14. Discard the supernatant and wash the precipitate with 70% ethanol. After air drying, the precipitate is resuspended in 20 g of DEPC-treated water.
Stage 4: Reverse Transcription PCR
Material
Buffers and Solutions
Dilute the storage solution in the appropriate proportion
10x Amplification Buffer
Chloroform
DEPC treated water
DTT (lmol/L)
dNTP solution at 1.25 mmol/L for each nucleotide concentration
Phenol
RNase inhibitor
Enzyme and Buffer
Bst XI
EcoR V
Mo-MLV reverse transcriptase
Taq DNA Polymerase
DNA uracil glycosylase (1 unit/ul) 
Gel
Agarose gel (1.5% m/V), prepared with TBE
Nucleic acids and oligonucleotides
Oligonucleotide primers [20 mmol/L, prepared in TE (pH 8.0)]. 
RNA was extracted from COS-7 cells transfected with control vector and recombinant vector, respectively (stage 3).
Medium
LB agar plates containing 50ug/ml ampicillin
Specialized equipment
Sampler tip for automatic micro-sampler
Microcentrifuge tubes (0.5 ml thin-walled tubes for PCR amplification, RNase-free)
Autosampler with Tip-Off Device
Thermal cycler with programmable reaction routines
Thermal cyclers without thermal caps. The use of mineral oil or sealing paraffin prevents evaporation of the PCR reaction mixture. Sealing paraffin not only prevents evaporation, but also has the effect of temporarily cesium-isolating the reaction components (e.g., primer and template) before the reaction mixture is heated. This prevents amplification at the start of the reaction due to non-specific binding of the primers to the template.
Water baths preset to 42°C, 55°C and 65°C
Additional reagents
The reagents required for step 14 of this program are listed in Chapter 1, Program 25.
Vectors and Strains
E. coli DH5α (or other strain that produces alpha complementation)
Vector pBluescript II (KS or SK) (Stratagene Corporation)
Methods
cDNA synthesis
1. Use SA2 oligonucleotide as a primer and cytoplasmic RNA obtained in step 3 as a template to prepare the first strand of cDNA.
Mix the following reagents in an RNase-free 0.5 ml centrifuge tube.
RNA 3ul
10x Xieng Khouang Buffer 2.5ul
dNTP solution (1.25 mmol/L each) 4ul
0.1mol/LDTT 1ul
3' primer (SA2,20umol/L) 1.25ul
DEPC treated water 11.25ul
Place the reaction mixture at 65°C for 5 min.
Important: To minimize the formation of secondary structures in the RNA, do not place the reaction mixture on ice.
Then add:
RNase Inhibitor 1ul
Mo-MLV reverse transcriptase (200 units) 1ul
Incubate at 42°C for 90 min.
2. Use oligonucleotide primers SA2 and SD6 simultaneously for limited synthesis of the second strand of cDNA. Mix the following reagents in a sterile thin-walled centrifuge tube:
Reverse transcription reaction mixture (from step 1) 12.5ul
1x Amplification Buffer 4ul
dNTP solution (1.25 mmol/L each) 6ul
Primer SA2 (20umol/L) 2ul
Primer SD6(20umol/L) 2.5ul
Taq DNA polymerase 1~2 units
Add DEPC treated water to 40ul
Also treat vector-only control as an important control for PCR analysis.
3. If the thermal cycler does not have a thermal lid, add 1 drop (~50ul) of light gravity mineral oil to the top of the reaction mixture. Alternatively, add sealing paraffin as for hot-start PCR. Place the reaction tube into the thermal cycler.
4. Perform the amplification reaction at the denaturation, denaturation and extension times and temperatures listed in the table below. 
Preparation of cDNA and vector for cloning
5. Add 30 units of BstXI to the PCR reaction product and 1 drop of low gravity mineral oil (if not added in the previous steps). The mixed reaction tube is placed in a warm bath at 55°C overnight.
BstXI digestion will greatly minimize empty vector splice products and false positives due to vector introns containing hidden splice sites. The vector plasmid pSPL3 contains multiple cryptic splice sites. Abnormal splice products caused by these sites generally represent a small fraction of the amplification product and are usually found only in empty vector reaction tubes, poor quality libraries constructed on pSPL3, or genomic DNA that does not contain transcripts that can be identified by this method.
6. Add another 20 units of BstXI to the reaction tube and place the tube in a 55BstXI warm bath for 2 to 3 hours.
7. Concurrently with the above steps, thoroughly digest 1ug of plasmid vector pBSII (KS or SK) with EcoR V.
Phenol extraction and ethanol precipitation were used to purify the DNA from the digested product, and the purified product was diluted to 2ng/ul with water.
8. Mix the following reagents in a 0.5 ml PCR amplification tube:
BstXI digested reverse transcription PCR product (step 6) 5-10ul
10X Amplification Buffer 4.5ul
dNTP (1.25 mmol/L per product) 7.5ul
Primer SADU(20umol/L) 2.5ul
Primer SDDU(20umol/L) 2.5ul
Taq DNA polymerase 10~20 units
Add H20 to 45ul
9. Mix the following reagents in another 0.5 ml PCR amplification tube:
EcoRV digested pBSII (KS or SK) (2ng/ul) 10ul
10x Amplification Buffer 10ul
dNTP (1.25 mmol/L per species) 16ul
Primer BSD-U (20umol/L) 5ul
Primer BSA-U(20umol/L) 5ul
Taq DNA Polymerase Unit 2~4 Units
Add H2O to 100ul
10. If the thermal cycler does not have a thermal cap, add 1 drop (~50ul) of light gravity mineral oil to the reaction mixture (steps 8 and 9). Alternatively, add a sealing paraffin pellet as for hot start PCR. Place the reaction tube into the thermal cycler.
11. Perform the amplification reaction with the denaturation, denaturation and extension parameters listed in the table below. 
12.1.5% agarose gel (TBE preparation) A small amount of PCR product is analyzed by electrophoresis to evaluate the quality of the reaction. The reaction product should be a typical diffuse band (Figure 11-19). 
Cloning of exon capture products
13. Add the following reagents to a small 0.5 ml centrifuge tube:
BstXI-cleaved cDNA (step 8) after
BstXI digested cDNA (step 8) 3ul of the product from the amplification reaction in step 11
EcoRV digested pBSⅡDNA (step 9) was amplified by the following reaction
Step 11: 1ul of the amplified product
10x amplification buffer 1ul
DNA uracil glycosylase (UDG, 1 unit/ul) 1ul
Add H2O to 10ul
Mix and incubate at 37°C for 30 min.
Important: In this reaction, DNA containing dU residues will be degraded by DNA uracil glycosylase, and recombinants will be formed between the digested plasmid with complementary ends and the amplified cDNA. To prevent nonspecific binding of the vector to the PCR product, do not place the tubes on ice after incubation at 37°C. If steps 14 and 15 cannot be performed immediately, continue to freeze the tubes at 37°C or -20°C. If Steps 14 and 15 cannot be performed immediately, continue to freeze the reaction tubes at 37°C or -20°C.
14. Convert 30-50 ul of DH5α broth into the entire UDG reaction mixture by the CaCl2 method.
15. Take 100-500ul of the transformed bacterial solution and spread it on LB agar plates containing 50ug/ml ampicillin and incubate at 37°C overnight.
Stage 5: Cloning analysis
Material
Buffers and solutions
Dilute the storage solution in the appropriate proportion.
10x Amplification Buffer
Enzyme and Buffer
TaqDNA Polymerase
Gel
Agarose gel (1.5% m/V),TAE preparation
See step 7.
Nucleic acids and oligonucleotides
dNTP solution, the concentration of all four nucleotides is 1.25 mmol/L
Exon capture products cloned into the vector pBSII
was prepared as a recombinant DH5a clone (see Stage 4).
Oligonucleotide primers [concentration 20 mmol/L dissolved in TE (pH 8.0) 
Medium
LB broth medium containing 50ug/ml ampicillin
LB broth medium with 30% glycerol (v/v)
Specialized equipment
96-well microtitre plate for PCR
Multi-Channel Sampler or Sampling Device (e.g. Dank or Scientific)
Thermal cyclers that fit into a 96-well microtitration plate
Additional reagents
The reagents needed for step 8 of this protocol are listed in Chapter 12, protocols 3, 4 or 5.
Methods
1. Add 100ul of LB broth medium containing 50ug/ml ampicillin to each well of a 96-well microtitre plate and add one independently grown clone of the transformants to each well (from Step 15 of Phase 4). Seal the plate with Parafilm and grow at 37°C for 3-4 h without shaking the plate.
2. Prepare a mixture for 100 PCR reactions by mixing the following components:
10X Amplification Buffer 250ul
dNTP solution (1.25 mmol/L each) 400ul
-20 oligonucleotide primer (20 mmol/L) 125ul
REV oligonucleotide primer (20 mmol/L) 125ul
H2O 1475ul
Taq DNA polymerase 75 units
Add 24ul of the above mixture to each well of a new 96-well PCR reaction plate.
3. Using a 96-well microdosing replicator, transfer the bacterial solution from the titration plate in step 1 to the 96-well PCR reaction plate containing the reaction mixture.
4, Add 1 drop (~50ul) of mineral oil to each well of the reaction solution if using a thermal cycler without a thermal cap. Alternatively, 1 solid paraffin pellet per well can be added in a similar way to the hot start method. Place the 96-well PCR plate in the thermal cycler.
5. Perform PCR amplification according to the denaturation, denaturation and extension times and temperatures in the following table 
6. Replicate the bacterial solution prepared in step 1 onto another titration plate using a multiwell spiker, shake overnight at 37°C, add 100ul of LB broth medium containing 30% glycerol to each well, seal with Parafilm membrane and freeze at -80°C.
7. Analyze the amplification products of each well in step 5 by electrophoresis on a 1.5% agarose gel.
The amplified bands shown by electrophoresis should be of different sizes, and it is not possible to determine the redundancy of the ETP on the basis of size, since the PCR products are concentrated at 120 bp.
8. Determine the sequence of each ETP (see Scheme 3, 4 or 5 in Chapter 12).
If the vector used for cloning is PBSIISK-sequencing with the T7 primer results in a sequence that starts at the 5' end of the ETP. The pSPU sequences flanking the ETP sequence are shown below: 