( ...)TripleMaster PCR System

1 Introduction 2 Precautions and warnings 3 Materials 3.1 Materials supplied with the kits 3.2 Materials required but not supplied 4 Enzyme concentration 5 Enzyme storage buffer composition 6 Storage and stability 7 Quality assurance 8 Buffer choice 9 Protocols 9.1 Long range PCR 9.2 High fidelity PCR of targets between 100 bp 10 kb 9.3 Amplification of GC-rich targets 10 Troubleshooting 10.1 No product 10.2 Little product on high background smear 10.3 Low yield 10.4 Non-specific product 11 Additional information 11.1 Reaction volume 11.2 Use of PCR additives and co-solvents with TripleMaster PCR System 11.3 Enzyme concentration 11.4 Concentrations of dNTPs and Mg²⁺ 11.5 Dilution of the TripleMaster Polymerase Mix 11.6 Fidelity 11.7 Cloning 11.8 Sequencing 11.9 Primer sequences for long targets

1 Introduction

The TripleMaster PCR System combines a powerful polymerase blend with an innovative two buffer system for efficient amplification of targets ranging from 100 bp up to 50 kb, including GC-rich targets and other difficult templates. The TripleMaster Polymerase Mix is a blend of thermostable DNA polymerases with a processivity-enhancing factor providing both an extremely high extension rate and maximal proofreading assisted fidelity.

Both buffers, the Tuning Buffer and the HighFidelity Buffer, of the TripleMaster PCR System have a unique zwitterionic formulation which improves pH-maintenance at high temperatures (7294C), thus reducing pH-driven template degradation to a minimum. The Tuning Buffer is designed for high fidelity long range PCR applications resulting in robust amplification of genomic targets >20 kb and episomal targets up to 50 kb without organic co-solvents. The HighFidelity Buffer is designed for high fidelity amplifications of smaller targets ranging from 100 bp to 10 kb with genomic templates and up to 15 kb with plasmid or phagemid DNA.

Control reagents are supplied with the TripleMaster PCR System Plus for the amplification of a 40 kb fragment.

Applications include amplification of:
very long targets (genomic > 20 kb; episomal up to 50 kb)
long targets (genomic 10 - 20 kb, episomal 10 - 40 kb)
150 bp 10 kb targets with high fidelity for cloning and sequencing purposes
rare and long cDNA from RT reactions
GC-rich and other difficult templates.

2 Precautions and warnings

Appropriate safety apparel such as lab coat, gloves, and eye protection should be worn. For more information, please consult the appropriate material safety data sheets which are available for this kit on line at www.eppendorf.com/msds

3 Materials

3.1 Materials supplied with the kits: Component 100 U
(50 - 100 reactions) 200 U
(100 - 200 reactions) 500 U
(250 500 reactions) TripleMaster Polymerase Mix (5U/l) 20 l 40 l 100 l 10x Tuning Buffer with Mg2+ 1.5 ml 1.5 ml 1.5 ml 10x HighFidelity Buffer with Mg2+ 1.5 ml 1.5 ml 2 x 1.5 ml 25 mM Magnesium Solution 1.5 ml 1.5 ml 1.5 ml Also available is the TripleMaster PCR System Plus. It includes the above components as well as: 20 l of Control DNA (10 ng/l) and 20 l of Control Primer Mix (10 pmol/l each), enough for 20 control reactions. 3.2 Materials required but not supplied

Template DNA
Primers
dNTPs*
Molecular Biology Grade Water*
*Products available through Eppendorf

4 Enzyme concentration

5 U/l*
*concentration refers to the amount of Taq DNA polymerase per l of the TripleMaster Polymerase Mix, although the specific activity (by nucleotide incorporation) of Taq in combination with the other enzymes is higher than the specific activity of pure Taq of the same concentration.

5 Enzyme storage buffer composition

TripleMaster Polymerase Mix: 20 mM Tris-HCl pH 8.0 (at 25C), 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 50 % glycerol, 0.5 % Tween 20, 0.5 % Igepal CA-630.

6 Storage and stability

Store all reagents of the TripleMaster PCR System at 20C in a constant-temperature freezer. If stored as recommended, the kit is stable at least until the expiration date stated on the label.

7 Quality assurance

Each lot of the TripleMaster PCR System is performance-tested in long range PCR. Routinely, TripleMaster PCR System is tested with genomic DNA using specific primers for the human tPA gene to obtain 24 and 27 kb products as well as with DNA and specific primers to amplify a 40 kb product.

8 Buffer choice

Choosing the appropriate reaction buffer is essential for obtaining optimal results with the TripleMaster PCR System. Use Table 1 as a guide for buffer selection in conjunction with the target size and template used. For targets ranging between 2 - 10 kb it is recommended to try both buffers and then choose the one with the best ratio of yield to specificity. For amplification of targets <= 500 bp or those with a low template concentration (< 10 ng/50 l of genomic DNA or < 500 pg/50 l of plasmid DNA) increase the final buffer concentration from 1x to 1.6x by adding 8 l of the 10x buffer to a 50 l assay. The resultant higher ionic strength of the reaction medium forces the TripleMaster Polymerase Mix to amplify short DNA fragments with a higher yield on small amounts of template DNA. Table 1: Buffer selection guide

9 Protocols

9.1 Long range PCR

Amplification with TripleMaster PCR System using the Tuning Buffer is robust and efficient for long targets up to 50 kb, even those where other enzymes fail.
General hints
- Use high molecular weight DNA templates only, stored at 28C. Avoid freezing the template DNA. Check the quality of the DNA; the average size must be > 50 kb.
- Use thin-walled 0.2 ml PCR tubes.
- Ensure that the 0.2 ml PCR tubes fit exactly into the thermal block of the cycler. Eppendorf 0.2 ml PCR tubes are recommended for the Eppendorf Mastercycler.
- Always prepare two master mixes and chill them on ice.
- Set up the complete reactions on ice.
- Mix the final reaction by gently pipetting up and down (three times). Oil overlay is not necessary for cyclers equipped with heated lids.
- Always use an elongation temperature of 68C and a denaturation temperature of 93C.
- Use a final dNTP concentration of 500 M for targets > 10 kb.
- Primers should have annealing temperatures above 60C.
- Do not apply hot start techniques such as Ampliwax.

Protocol for long range PCR

Prepare two Mastermixes and keep them on ice. Mastermix 1 contains primers and template. Mastermix 2 contains enzyme, dNTPs and buffer. Combine Mastermixes 1 and 2 immediately prior to commencing cycling in order to avoid primer/template degradation by 35 exonuclease activity and unspecific primer extension at lower temperatures.

1. Thaw the reagents listed in Table 2 carefully and keep on ice.
2. Make sure reagents are thoroughly thawed. Mix each reagent and briefly centrifuge before use.
3. If various amounts of the TripleMaster Polymerase Mix are to be tested, prepare an enzyme dilution in 1x Tuning Buffer as described in the Appendix.
4. Set up Mastermix 1 in a sterile microcentrifuge tube, mix well, centrifuge and place on ice (see Table 2).
5. Set up Mastermix 2 in a seperate sterile microcentrifuge tube, mix well, centrifuge and place on ice (see Table 2).
6. Just before cycling, pipette 10 l of Mastermix 1 and then 40 l of Mastermix 2 into a 0.2 ml PCR tube on ice. Mix gently by pipetting 3-4 times up and down. Do not vortex and/or centrifuge!
7. Place immediately into a thermal cycler which is pre-heated at 93C and start cycling. Examples of cycling profiles for various long targets are given for the Eppendorf Mastercycler (Table 4). For customer-specific targets, only the annealing temperature should be adapted. By changing the time of the elongation step the product yield can be optimized following the rules given in Table 4 for different target sizes and template complexity.
8. Analyze large amplification products on a 0.4 % agarose gel preferably in TAE-buffer using appropriate DNA markers. For separation of products > 25 kb in a standard electrophoresis chamber, a 12-hour run at 40 V is recommended. Generally, efficient separation of very large DNA fragments is achieved only by inverse or pulse-field
   electrophoresis.

Table 2: Mastermix composition for long range PCR ¹ components included in the TripleMaster PCR System Plus
² some applications may require an extra addition of Magnesium Solution (supplied) to set up a final concentration >2.5 mM, e.g. when DNA amounts >0.5 g are used.

Cycling program for long range PCR

The complete program for the 40 kb control amplification included in the TripleMaster PCR System Plus on the Eppendorf Mastercycler is given in Table 3. This data should be used as a guide for customized adaptation of the cycling program to an appropriate primer/template system and cycler. Program parameters printed in bold should be kept invariable whereas other parameters are variable for primer/template-specific adaptation. Optimized values for variable program parameters on Eppendorf Mastercycler for a number of genomic and episomal targets are given in Table 4.

Table 3: Program parameters optimized for the 40 kb control PCR * Time increment of 20 seconds; for each elongation step the time is extended by 20 seconds (21 min 20 sec in 11th cycle; 21 min 40 sec in 12th cycle, etc).

Table 4: Variable cycling parameters optimized for various long targets 9.2 High fidelity PCR of targets between 100 bp 10 kb

For targets < 2 kb use the TripleMaster Polymerase Mix preferably in combination with the HighFidelity Buffer. For targets 2 kb < 10 kb test both the Tuning and HighFidelity Buffer and choose the combination that provides a better yield and/or specificity. If amplifying fragments < 500 bp using a low concentration of template DNA, increase the final buffer concentration in the PCR assay from 1x to 1.6x (8 l of 10x buffer in a 50 l reaction).

General hints

The fidelity of DNA amplification depends on the type of polymerase used and other factors, such as dNTP and Mg2+ concentration, template complexity and base composition, cycle number and pH of the reaction. The TripleMaster Polymerase Mix is optimized for high fidelity. Changing other reaction conditions, instead of changing the DNA polymerase, can further increase fidelity. Rules for optimization of general reaction conditions to achieve maximum fidelity are provided below:

- Start with 200 M of dNTP concentration. For smaller targets up to 2 kb try final concentrations of 80, 100, 120 and 150 M to further increase the PCR fidelity. - Run only as many cycles as necessary for obtaining enough product: 30 cycles for single-copy genomic targets, 25-28 cycles for plasmid/phage targets or multicopy genomic targets. - Increase initial template concentration to obtain more product. - Use high quality dNTPs only. dNTP solutions which are contaminated by chemically altered bases, pyrophosphate, salts, rNTPs, dNDPs and dNMPs lead to misincorporation, mispairing, strand termination, nucleotide modification and excision and inhibit the DNA polymerases. dNTP contamination has a direct impact on DNA amplification fidelity. - Avoid excess of enzyme in amplification reactions. For high fidelity PCR use 0.51.0 U per 50 l (see Table 5). Empirically determine the lowest possible amount of enzyme to obtain enough product and sensitivity for an individual application. - Variable reaction volumes (20, 25, 50, or 100 l) and tube formats (0.2 or 0.5 ml) can be used. Adjust the amount of enzyme per reaction respectively (see Table 5). Note: Adjusting reaction conditions for maximal PCR fidelity always leads to a reduction of product yield!

Because of the wide target size range for high fidelity PCR, optimal reaction parameters (incubation times, temperatures, concentrations of template DNA, primer, Mg2+ and enzyme mix) vary and must be determined for each primer/template pair individually.

Protocol for high fidelity PCR

Prepare two Mastermixes and keep them on ice. Mastermix 1 contains primers and template. Mastermix 2 contains enzyme, dNTPs and buffer. Combine Mastermixes 1 and 2 immediately prior to commencing cycling in order to avoid primer/template degradation by 35 exonuclease activity and non-specific primer extension at lower temperatures.

1. Thaw the reagents listed in Table 5 carefully and keep on ice.
2. Make sure reagents are thoroughly thawed. Mix each reagent and briefly centrifuge before use.
3. Prepare an enzyme dilution in 1x HighFidelity Buffer as described in the appendix, if variable amounts of enzyme units will be tested.
4. Set up Mastermix 1 in a sterile microcentrifuge tube, mix well, centrifuge and place on ice (see Table 5).
5. Set up Mastermix 2 in a seperate sterile microcentrifuge tube, mix well, centrifuge and place on ice (see Table 5).
6. Just before cycling, pipette Mastermix 1 and Mastermix 2 into a separate PCR tube on ice. Mix thoroughly (vortex) and centrifuge.
7. Place immediately into a thermal cycler pre-heated at 94C and start cycling. Example of cycling profile is given for the Eppendorf Mastercycler (see Table 6).
8. Analyze amplification products on a 0.61.5% agarose gel using appropriate DNA markers.

Table 5: Mastermix composition for high fidelity PCR ¹ some applications may require an extra addition of Magnesium Solution (supplied) to set up a final concentration >2.5 mM.
² increase final buffer conc. (1.6x) for targets < 500 bp or when using a low concentration of template DNA per
reaction (< 10 ng genomic DNA or < 0.5 ng plasmid DNA)

Cycling program for high fidelity PCR

A standard program which can be adapted to an appropriate primer/template system is shown in Table 6. The data of Table 7 may be used as a guide for the adaptation of individual PCR reactions.

Table 6: Program for high fidelity PCR with Eppendorf Mastercycler ¹ Number of cycles depends on product yield required, template concentration and complexity.
² The annealing temperature depends on the melting temperatures of the primer set used.
³ This elongation temperature is recommended for long targets >5 kb to reduce the temperature-driven damage of template DNA.
⁴ The elongation time depends on fragment length and type of thermal cycler. Elongation times with the Eppendorf astercycler for various fragment sizes are given in Table 7. These values can also be transferred to other Peltier-driven cyclers.

Table 7: Target size and elongation time ¹ The elongation time required for targets with an unusual (non-random) nucleotide base composition, repeats or high GC-content is longer than for standard targets of the same size. In those cases, work at the upper limit of time range given.

9.3 Amplification of GC-rich targets

With the TripleMaster PCR System, DNA sequences with a biased nucleotide composition (GC-content >70 %; homopolymeric stretches), repeats and inverted repeats can be amplified.

General hints

The reaction parameters, especially for GC-rich targets, may require some modification:
- Test which reaction buffer performs best with the appropriate target (Tuning Buffer versus HighFidelity Buffer) - Increase the temperature for template denaturation to 98C (not feasible for long targets >5 kb). - Use the upper limit of the time range given in Table 7 (processivity and extension rate of polymerases on difficult templates is slower). - Work at higher dNTP concentrations (300-500 M) than given in the protocol for high fidelity PCR. - For extremely GC-rich targets (>70 %) use GC-destabilizing co-solvents in combination with the Tuning or HighFidelity Buffer (DMSO: 2-8 %; glycerol: 2-5 %; betaine: 0.5- 2.5 M; trimethylammonium hydrochloride: 50-100 M; acetamide: 1-5 %). Reduce annealing temperature by 2C decrements with co-solvents. - Increase the denaturation temperature gradually up to 98C using the gradient function of the Mastercycler gradient, but restrict the denaturation time in each cycle to maximal 10 seconds. - Introduce a special pre-amplification denaturation step at 98C with the buffer/template/primer mix containing DMSO (see step 4 in protocol below and table 8). Protocol for GC-rich targets

1. Thaw the reagents listed in Table 8 carefully and keep on ice.
2. Make sure reagents are thoroughly thawed. Mix each reagent and briefly centrifuge before use.
3. Set up Mastermix 1 (see Table 8) containing buffer, primer, template DNA and DMSO (optional) in a sterile microcentrifuge tube, mix well, centrifuge and heat for 30 seconds at 98C.
4. Quickly spin down (2 sec) Mastermix 1 and immediately place on ice.
5. Prepare Mastermix 2 (see Table 8) in a separate sterile microcentrifuge tube, mix well, centrifuge and place on ice.
6. Just before cycling, pipette Mastermix 1 and then Mastermix 2 into a separate PCR tube on ice. Mix quickly but thoroughly, (vortex) and centrifuge.
7. Place immediately into a thermal cycler that is pre-heated at 98C (use the "Hold" step for this) and start cycling.
8. Analyze amplification products on a 0.6-1.5 % agarose gel using appropriate DNA markers.

Table 8: Mastermix conditions for GC-rich PCR ¹ increase final buffer conc. (1.6x) for targets < 500 bp or when using a low concentration of template DNA per reaction (< 10 ng genomic DNA or < 0.5 ng plasmid DNA)
² increase dNTP concentration for GC-rich targets

Table 9: Cycling program for GC-rich targets ¹ Difficult targets require more cycles (and template DNA) for efficient amplification. Start with 35 cycles, if necessary, increase up to 45 cycles.
² Determine the optimal annealing temperature empirically.
³Optimize elongation temperature. Increasing temperature >72C is not recommended for targets >5 kb due to temperature-driven damage of template DNA.
⁴ The elongation time depends on fragment length and template complexity. Use the upper values for each fragment size range given in Table 7 to amplify GC-rich targets. Further increase the elongation time if the product yield is still low or a background smear occurs.

10 Troubleshooting

Follow the manual as written!

10.1 No product

- Check the quality of primers and template. - Check the annealing and denaturation temperatures (especially for GC-rich targets). - Increase number of cycles (by steps of 5). - Increase template DNA by 50 ng increments. - Increase enzyme concentration (up to 2.5 U per 50 l). - Check the fit of the PCR tubes to the appropriate cycler. Bad contact affects temperature transfer. Use only consumables recommended by the cycler manufacturer. - Prevent air bubbles from becoming trapped after mixing the reaction Mastermixes. Air bubbles prevent homogenous temperature distribution throughout the reaction volume. - Check the fit of PCR tube caps or sealing foils on PCR plates. Long-range PCR is sensitive to evaporation. 10.2 Little product on high background smear

- Reduce number of cycles by steps of 2. - Reduce template DNA by 50 ng decrements. - Increase the time of the elongation step (But do not increase the time extension per cycle in the long range PCR program). - Check the ratio of magnesium ions/dNTP. A final dNTP concentration at 500 M requires 2.5 mM Mg²⁺. - Reduce the amount of enzyme in 0.2 U decrements. - Increase the concentration of magnesium ions up to 3.5 mM in 0.25 increments if more than 500 ng template DNA, or more than 500 M dNTPs are used. 10.3 Low yield

- Choose the appropriate reaction buffer (see Table 1): Tuning Buffer for 240 kb PCR on genomic DNA and 1050 kb PCR on plasmid/phagemid DNA.
HighFidelity Buffer for 0.110 kb PCR on genomic DNA and plasmid/phagemid DNA. - Check the Tuning Buffer versus HighFidelity Buffer for GC-rich and other complex targets of any size between 210 kb. - Increase the final buffer concentration in the reaction assay to 1.6x (4 l of 10x HighFidelity or Tuning Buffer in 25 l or 8 l in 50 l, respectively) for targets < 500 bp or when using a low amount of template DNA. - Increase the number of cycles (especially for complex genomic targets and GC-rich targets). - Increase the amount of template DNA per reaction by 50 ng increments. - Increase the amount of enzyme (up to 2.5 U per 50 l). - Increase the magnesium ions (up to 3.5 mM).
10.4 Non-specific product

- Try higher annealing temperatures. - Reduce the annealing time down to 8 seconds in 2 seconds decrements. - Reduce time of the elongation step (see Table 7). - Check Tuning Buffer versus HighFidelity Buffer (especially for targets < 2 kb). - Decrease the enzyme amount per reaction in 0.2 U decrements. - Reduce the amount of template DNA. - Reduce the number of cycles by steps of 2. - Add 12 % DMSO to a 50 l reaction. - Design new/longer primers.

11 Additional information

11.1 Reaction volume

The use of 0.2 ml thin-walled PCR tubes is recommended for long range PCR and other difficult PCR applications with a reaction volume of 50 l, and for high fidelity PCR applications of 2025 l.

11.2 Use of PCR additives and co-solvents with TripleMaster PCR System

In most cases, the TripleMaster PCR System produces good results without any additive, with either the Tuning or HighFidelity Buffer. For very GC-rich (>70%) templates or templates containing complex secondary structure, the addition of iso-stabilizing cosolvents such as betaine (0.53 M final concentration), DMSO (25 % final), glycerol (25%), trimethylamine N-oxide (10100 M) or trimethyleamine hydrochloride (10100 M) may improve yield and specificity of PCR. However, the use of destabilizing co-solvents substantially reduces the PCR fidelity and requires a re-optimization of the annealing temperature. The TaqMaster PCR Enhancer, available separately, can be used to stabilize the enzyme for elevated denaturation temperatures (up to 98C) during amplification of GCrich targets. This approach is not feasible in long range PCR.

11.3 Enzyme concentration

Long range applications: 2 U = 0.4 l Other applications: 0.52.5 U = 0.10.5 l 11.4 Concentrations of dNTPs and Mg²⁺

Both buffers of the TripleMaster PCR System provide a final Mg concentration of 2.5 mM which is ideal for most applications. An addition of 25 mM Magnesium Solution to the PCR reactions (in increments of 0.2 mM) is necessary only if the template concentration exceeds 500 ng per 50 l PCR volume and/or the dNTP concentration is higher than 500 M.

dNTP / Mg²⁺ Long range PCR: 500 M / 2.5 mM High fidelity PCR: 200 M / 2.5 mM (optional 80150 M/2.5 mM) PCR of GC-rich targets: 250500 M / 2.5 mM 11.5 Dilution of the TripleMaster Polymerase Mix

Prepare a 1:10 enzyme dilution as follows:
Add 32 l Molecular Biology Grade Water and 4 l of the corresponding 10x reaction buffer to a sterile microcentrifuge tube, mix well and spin down. Then add 4 l of the TripleMaster Polymerase Mix, mix thoroughly (glycerol solution) and spin down quickly. Keep the enzyme dilution on ice until use. It is stable for several hours. Do not store overnight!

11.6 Fidelity

In combination with both reaction buffers, the TripleMaster Polymerase Mix exhibits a 45 fold higher fidelity (2.3 x 10^-6) than Taq DNA polymerase (12.5 x 10^-6). The TripleMaster PCR System keeps the same high fidelity for long range amplification of large genomic targets (> 20 kb) as for amplification of short targets, because it circumvents the use of fidelityreducing co-solvents such as DMSO. Note: The addition of 5 % DMSO to the PCR causes a reduction of polymerase fidelity by more than 50 %. The additon of 10 % DMSO reduces the fidelity of proofreading polymerase blends to the level of Taq DNA polymerase alone.

11.7 Cloning

PCR products generated by the TripleMaster PCR System contain a mixture of fragments with blunt ends and 3-single nucleotide overhangs analogous to PCR products amplified by Taq DNA polymerase. TAcloning is therefore recommended. For blunt-end cloning, the fragments need to be polished by Klenow enzyme or T4 DNA polymerase.

11.8 Sequencing

Sequencing with the TripleMaster Polymerase Mix is not recommended due to the inherent 35 exonuclease activity.

11.9 Primer sequences for long targets

universal forward primer (in the control primer mix) :
5-CTGATGAGTTCGTGTCCGTACAACTGGCGTAATC-3 reverse primers: 40 kb: 5-TAATGCAAACTACGCGCCCTCGTATCACATGG-3 (in the control primer mix) 35 kb: 5-ATTATGTCGGTGATACTTCGTCGCTGTCTC-3 30 kb: 5-GAAAGTTATCGCTAGTCAGTGGCCTGAAGAGACG-3 20 kb: 5-GTGCACCATGCAACATGAATAACAGTGGGTTATC-3 10 kb: 5-ATACGCTGTATTCAGCAACACCGTCAGGAACACG-3 universal forward primer for human tPA gene: 5-CCTTCACTGTCTGCCTAACTCCTTCGTGTGTTCC-3 reverse primer for human tPA gene: 15 kb: 5- ACTGTGCTTCCTGACCCATGGCAGAAGCGCCTTC-3 18 kb: 5-GCAGGGGTGCTGCAGAACTCTGAGCTGTACTTCC-3 22 kb: 5-GATGCGAAACTGAGGCTGGCTGTACTGTCTC-3 24 kb: 5-TGTCTCCAGCACACAGCATGTTGTCGGTGAC-3 27 kb: 5-CAAAGTCATGCGGCCATCGTTCAGACACACC-3
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