TITLE: GGAssmbler Library Construction
AUTHORS: Shlomo Yakir Hoch, Ravit Netzer, Karen Hakeny, Sarel J Fleishman

[DESCRIPTION]
This protocol describes methods for GGAssembler.

[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.

[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:
DNAAMOUNTDNA Genomic1 ng–1 µgPlasmid or Viral1 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 ).

[STEPS]
SECTION: Amplify constant fragments
1. Set up the following reaction on ice:
 

 

SECTION: Amplify constant fragments
2. Gently mix the reaction.
SECTION: Amplify constant fragments
3. Collect all liquid to the bottom of the tube by a quick spin if necessary and overlay the sample with mineral oil if using a PCR machine without a heated lid. 
SECTION: Amplify constant fragments
4. Quickly transfer PCR tubes to a PCR machine preheated to the denaturation temperature (98 °C) and begin thermocycling. 

Thermocycling Conditions for a Routine PCR:
STEPTEMPTIMEInitial Denaturation98°C30 seconds25–35 Cycles98°C5–10 seconds*50–72°C10–30 seconds72°C20–30 seconds/kbFinal Extension72°C2 minutesHold4–10°C 

 
 
SECTION: Amplify constant fragments
5. (IF REQUIRED) Digest the lead gene templates with DpnI.

SECTION: Amplify constant fragments
6. Quantify DNA concentration by Qubit.
SECTION: Amplify constant fragments
7. Measure DNA length using TapeStation.
SECTION: Variable fragments fill in
8. Follow the steps for Amplify constant fragments (steps 1-7).
SECTION: Variable fragments fill in
9. Combine variable fragments of the same segment in equal concentration.
SECTION: Golden Gate Assembly
10. Set up 25 µl assembly reactions as follows: 
  REAGENTSASSEMBLY REACTIONDNA inserts 100 ng/ul each1 µl (~ 100 ng) each, (up tp 19.5 µl)T4 DNA Ligase Buffer (NEB #B0202) (10X)2.5 μlT4 DNA Ligase (NEB #M0202), 2000 U/µl0.5 μl (1000 units)BsaI-HFv2 (NEB #R3733), 20 U/µl1.5 μl (30 units)Nuclease-free H2O (NEB #B1500)to 25 µl

 

SECTION: Golden Gate Assembly
11. Mix gently by pipetting up and down 4 times.
SECTION: Golden Gate Assembly
12. Briefly microcentrifuge (1 second) to bring material to the bottom of tube.
SECTION: Golden Gate Assembly
13. Transfer to thermocycler and program as follows: (5 min 37ºC → 5 min 16ºC) x 30 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.
SECTION: Golden Gate Assembly Cleanup
14. 
 

Perform Oligonucleotide Cleanup (https://dx.doi.org/10.17504/protocols.io.bg9sjz6e) or follow a size selection protocol.
SECTION: Amplify Golden Gate Assembly Prodcuts
15. For 1x reaction set up the following reaction on ice:
 
 

   Component25 µl ReactionFinal ConcentrationQ5 High-Fidelity 2X Master Mix12.5 µl1X10 µM Forward Primer1.25 µl0.5 µM10 µM Reverse Primer1.25 µl0.5 µMTemplate DNA5 µlNuclease-Free Waterto 25 µl

 
 
SECTION: Amplify Golden Gate Assembly Prodcuts
16. Gently mix the reaction.
SECTION: Amplify Golden Gate Assembly Prodcuts
17. Collect all liquid to the bottom of the tube by a quick spin if necessary and overlay the sample with mineral oil if using a PCR machine without a heated lid. 
SECTION: Amplify Golden Gate Assembly Prodcuts
18. Quickly transfer PCR tubes to a PCR machine preheated to the denaturation temperature (98 °C) and begin thermocycling. 

Thermocycling Conditions for a Routine PCR:
STEPTEMPTIMEInitial Denaturation98°C30 seconds25–35 Cycles98°C5–10 seconds*50–72°C10–30 seconds72°C20–30 seconds/kbFinal Extension72°C2 minutesHold4–10°C 

 
 
SECTION: Golden Gate Assembly Amplification Cleanup
19. 
 

Perform Oligonucleotide Cleanup (https://dx.doi.org/10.17504/protocols.io.bg9sjz6e) or follow a size selection protocol.

SECTION: Golden Gate Assembly Amplification Quantification
20. Quantify DNA concentration by Qubit.
SECTION: Golden Gate Assembly Amplification Quantification
21. Measure DNA length using TapeStation.