Dec 18, 2023
Version 3
GGAssmbler Library Construction V.3
Version 1 is forked from PCR with Q5® Polymerase (M0491)
- Shlomo Yakir Hoch1,
- Ravit Netzer1,2,
- Lucas Krauss1,2,
- Karen Hakeny1,
- Sarel J Fleishman1,2
- 1Weizmann Institute of Science;
- 2Scala Biodesign LTD
Protocol Citation: Shlomo Yakir Hoch, Ravit Netzer, Lucas Krauss, Karen Hakeny, Sarel J Fleishman 2023. GGAssmbler Library Construction. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbxqkolpk/v3Version created by Shlomo Yakir Hoch
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 17, 2023
Last Modified: December 18, 2023
Protocol Integer ID: 92429
Funders Acknowledgement:
Volkswagen Foundation
Grant ID: 94747
Israel Science Foundation
Grant ID: 1844
European Research Council through a Consolidator Award
Grant ID: 815379
Dr. Barry Sherman Institute for Medicinal Chemistry
donation in memory of Sam Switzer
Abstract
This protocol describes methods for GGAssembler.
Guidelines
Please note that protocols with Q5® High-Fidelity DNA Polymerase may differ from protocols with other polymerases. Conditions recommended below should be used for optimal performance.
Reaction Setup:
We recommend assembling all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (98°C). All components should be mixed prior to use. Q5 High-Fidelity DNA Polymerase may be diluted in 1X Q5 Reaction Buffer just prior to use in order to reduce pipetting errors.
General Guidelines:
1. Template:
Use of high quality, purified DNA templates greatly enhances the success of PCR. Recommended amounts of DNA template for a 50 µl reaction are as follows:
| A | B | |
| DNA Genomic | 1 ng–1 µg | |
| Plasmid or Viral | 1 pg–1 ng |
2. Primers:
Oligonucleotide primers are generally 20–40 nucleotides in length and ideally have a GC content of 40–60%. Computer programs such as Primer3 can be used to design or analyze primers. The best results are typically seen when using each primer at a final concentration of 0.5 µM in the reaction.
3. Mg++ and additives:
Mg++ concentration of 2.0 mM is optimal for most PCR products generated with Q5 High-Fidelity DNA Polymerase. When used at a final concentration of 1X, the Q5 Reaction Buffer provides the optimal Mg++ concentration.
Amplification of some difficult targets, like GC-rich sequences, may be improved by the addition of 1X Q5 High GC Enhancer. The Q5 High GC Enhancer is not a buffer and should not be used alone. It should be added only to reactions with the Q5 Reaction Buffer when other conditions have failed.
4. Deoxynucleotides:
The final concentration of dNTPs is typically 200 μM of each deoxynucleotide. Q5 High-Fidelity DNA Polymerase cannot incorporate dUTP and is not recommended for use with uracil-containing primers or templates.
5. Q5 High-Fidelity DNA Polymerase concentration:
We generally recommend using Q5 High-Fidelity DNA Polymerase at a final concentration of 20 units/ml (1.0 unit/50 μl reaction). However, the optimal concentration of Q5 High-Fidelity DNA Polymerase may vary from 10–40 units/ml (0.5–2 units/50 μl reaction) depending on amplicon length and difficulty. Do not exceed 2 units/50 μl reaction, especially for amplicons longer than 5 kb.
6. Buffers:
The 5X Q5 Reaction Buffer provided with the enzyme is recommended as the first-choice buffer for robust, high-fidelity amplification. For difficult amplicons, such as GC-rich templates or those with secondary structure, the addition of the Q5 High GC Enhancer can improve reaction performance. The 5X Q5 Reaction Buffer is detergent-free and contains 2.0 mM Mg++ at the final (1X) concentration.
7. Denaturation:
An initial denaturation of 30 seconds at 98°C is sufficient for most amplicons from pure DNA templates. Longer denaturation times can be used (up to 3 minutes) for templates that require it.
During thermocycling, the denaturation step should be kept to a minimum. Typically, a 5–10 second denaturation at 98°C is recommended for most templates.
8. Annealing:
Optimal annealing temperatures for Q5 High-Fidelity DNA Polymerase tend to be higher than for other PCR polymerases. The NEB Tm Calculator should be used to determine the annealing temperature when using this enzyme. Typically, use a 10–30 second annealing step at 3°C above the Tm of the lower Tm primer. A temperature gradient can also be used to optimize the annealing temperature for each primer pair.
For high Tm primer pairs, two-step cycling without a separate annealing step can be used (see note 11).
9. Extension:
The recommended extension temperature is 72°C. Extension times are generally 20–30 seconds per kb for complex, genomic samples, but can be reduced to 10 seconds per kb for simple templates (plasmid, E. coli, etc.) or complex templates < 1 kb. Extension time can be increased to 40 seconds per kb for cDNA or long, complex templates, if necessary.
A final extension of 2 minutes at 72°C is recommended.
10. Cycle number:
Generally, 25–35 cycles yield sufficient product. For genomic amplicons, 30-35 cycles are recommended.
11. 2-step PCR:
When primers with annealing temperatures ≥ 72°C are used, a 2-step thermocycling protocol (combining annealing and extension into one step) is possible.
12. Amplification of long products:
When amplifying products > 6 kb, it is often helpful to increase the extension time to 40–50 seconds/kb.
13. PCR product:
The PCR products generated using Q5 High-Fidelity DNA Polymerase have blunt ends. If cloning is the next step, then blunt-end cloning is recommended. If T/A-cloning is preferred, the DNA should be purified prior to A-addition, as Q5 High-Fidelity DNA Polymerase will degrade any overhangs generated.
Addition of an untemplated -dA can be done with Taq DNA Polymerase (NEB #M0267 ) or Klenow exo– (NEB #M0212 ).
Materials
Materials
Q5 High-Fidelity DNA Polymerase - 100 unitsNew England BiolabsCatalog #M0491S
DpnI - 5,000 unitsNew England BiolabsCatalog #R0176L
T4 DNA Ligase Reaction Buffer - 6.0 mlNew England BiolabsCatalog #B0202S
T4 DNA Ligase - 20,000 unitsNew England BiolabsCatalog #M0202S
BsaI-HFv2New England BiolabsCatalog #
R3733S
Nuclease-free Water - 25 mlNew England BiolabsCatalog #B1500S
Monarch® PCR & DNA Cleanup Kit (5 μg)New England BiolabsCatalog #T1030
Equipment
Equipment
new equipment
NAME
Qubit 2.0 Fluorometer instrument
BRAND
Q33226
SKU
with Qubit RNA HS Assays
SPECIFICATIONS
Equipment
4200 TapeStation System
NAME
Electrophoresis tool for DNA and RNA sample quality control.
TYPE
TapeStation Instruments
BRAND
G2991AA
SKU
LINK
Safety warnings
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards.
Before start
Please note that protocols with Q5® High-Fidelity DNA Polymerase may differ from protocols with other polymerases. Conditions recommended below should be used for optimal performance.
Amplify constant fragments
Amplify constant fragments
Set up the following reaction On ice :
Note
All components should be mixed prior to use. Q5 High-Fidelity DNA Polymerase may be diluted in 1X Q5 Reaction Buffer just prior to use in order to reduce pipetting errors.
| A | B | C | |
| COMPONENT | 25 µl REACTION | FINAL CONCENTRATION | |
| 5X Q5 Reaction Buffer | 5 µl | 1X | |
| 10 mM dNTPs | 0.5 µl | 200 µM | |
| 10 µM Forward Primer | 1.25 µl | 0.5 µM | |
| 10 µM Reverse Primer | 1.25 µl | 0.5 µM | |
| Template DNA | variable | ~ 5 ng | |
| Q5 High-Fidelity DNA Polymerase | 0.25 µl | 0.02 U/µl | |
| 5X Q5 High GC Enhancer (optional) | (5 µl) | (1X) | |
| Nuclease-Free Water | to 25 µl |
Gently mix the reaction.
Collect all liquid to the bottom of the tube by a quick spin if necessary.
Quickly transfer PCR tubes to a PCR machine preheated to the denaturation temperature (98 °C ) and begin thermocycling.
Thermocycling Conditions for a Routine PCR:
| A | B | C | |
| STEP | TEMP | TIME | |
| Initial Denaturation | 98°C | 30 seconds | |
| 25–35 Cycles | 98°C | 5–10 seconds | |
| *50–72°C | 10–30 seconds | ||
| 72°C | 20–30 seconds/kb | ||
| Final Extension | 72°C | 2 minutes | |
| Hold | 4–10°C |
Digest the lead gene templates with DpnI at 37°C for 2 hours. Heat-inactivate at 80°C for 20 minutes.
Quantify DNA concentration by Qubit Fluorometer (Qubit 2.0 Fluorometer, ThermoFisher Scientific).
Measure DNA length using TapeStation (Agilent 2200 TapeStation).
Variable fragments fill in
Variable fragments fill in
Follow the steps for Amplify constant fragments (steps 1-9), skipping step 5.
Combine variable fragments of the same segment in equal concentration.
Golden Gate Assembly
Golden Gate Assembly
Set up 25 µl assembly reactions as follows:
| A | B | |
| REAGENTS | ASSEMBLY REACTION | |
| DNA inserts 100 ng/ul each | 1 µl (~ 100 ng) each, (up to 39 µl) | |
| T4 DNA Ligase Buffer (NEB #B0202) (10X) | 5 μl | |
| T4 DNA Ligase (NEB #M0202), 2000 U/µl | 1 μl (2000 units) | |
| BsaI-HFv2 (NEB #R3733), 20 U/µl | 3 μl (60 units) | |
| Nuclease-free H2O (NEB #B1500) | to 50 µl |
Mix gently by pipetting up and down 4 times.
Briefly microcentrifuge (1 second) to bring material to the bottom of tube.
Transfer to thermocycler and program as follows: (5 min 37ºC → 10 min 16ºC) x 60 cycles followed by 5 minutes 60ºC. If reactions are done overnight, add a 4ºC terminal hold to the protocol, but repeat the final 5 minutes 60ºC step the next day before the transformations.
Golden Gate Assembly Cleanup
Golden Gate Assembly Cleanup
Perform PCR DNA Cleanup (https://dx.doi.org/10.17504/protocols.io.bg9sjz6e) or follow a size selection protocol.
Amplify Golden Gate Assembly Prodcuts
Amplify Golden Gate Assembly Prodcuts
For 1x reaction set up the following reaction On ice :
Note
Using GG assembly products in concentration of 3-5ng/µl is highly recommended.
| A | B | C | |
| Component | 25 µl Reaction | Final Concentration | |
| Q5 High-Fidelity 2X Master Mix | 12.5 µl | 1X | |
| 10 µM Forward Primer | 1.25 µl | 0.5 µM | |
| 10 µM Reverse Primer | 1.25 µl | 0.5 µM | |
| GG assembly products | 5 µl | ||
| Nuclease-Free Water | to 25 µl |
Gently mix the reaction.
Collect all liquid to the bottom of the tube by a quick spin if necessary.
Quickly transfer PCR tubes to a PCR machine preheated to the denaturation temperature (98 °C ) and begin thermocycling.
Thermocycling Conditions for a Routine PCR:
| A | B | C | |
| STEP | TEMP | TIME | |
| Initial Denaturation | 98°C | 30 seconds | |
| 25–35 Cycles | 98°C | 5–10 seconds | |
| *50–72°C | 10–30 seconds | ||
| 72°C | 10 seconds/kb | ||
| Final Extension | 72°C | 2 minutes | |
| Hold | 4–10°C |
Golden Gate Assembly Amplification Cleanup
Golden Gate Assembly Amplification Cleanup
Note
This step is optional but HIGHLY recommended.
Perform Oligonucleotide Cleanup (https://dx.doi.org/10.17504/protocols.io.bg9sjz6e) or follow a size selection protocol.
Golden Gate Assembly Amplification Quantification
Golden Gate Assembly Amplification Quantification
Quantify DNA concentration by Qubit.
Measure DNA length using TapeStation.
Protocol references
24-Fragment Golden Gate Assembly using BsaI-HF‱v2 (NEB #R3733) https://www.neb.com/en/protocols/2021/01/11/24-fragment-golden-gate-assembly-using-bsai-hfv2