Protocol

1. Resuspend oligos to make a 100 uM stock and then 10 uM dilutions that combine oligo pairs

• Read nmol amount of oligo from tube label

• For every nmol, add 10 ul water

• Vortex or flick tube and wait several minutes for the oligo to go into solution

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2. In a separate tube combine 80 ul water and 10 ul of each 100 uM oligo in a pair to make 10 uM paired dilutions

• Oligo pairs that will be annealed as duplexes are named with the format X_s1 and X_s2. As an example, for X=T500:

• Oligo pairs that will be used together as PCR primers together are named with the format XGolgat_1F and X_Golgat##R or XY_Golgat_1F and X_Y'_Golgat##R.

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3. Prepare Golden Gate annealed duplex fragments.

• In separate PCR tubes, mix the reagents

• Incubate mix at 37C for 1 hr

• Heat mixtures at 95C in thermocycler for 5 min and gradually cool to RT for 1 hr

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4. Prepare the other Golden Gate fragments by PCR from plasmid X in primer pair X_Y_Golgat.

• In separate PCR tubes, mix the 50 ul PCR reactions

• Run the PCR program in your thermocycler with the lid set to 105 C

• Run finished PCR reaction on a 1% agarose TAE gel (see protocol homework 2)

• Purify the correct-sized band with a gel extraction kit (see kit manufacturer’s protocol)

 

5. Combine and assemble the Golden Gate fragments

• For each fragment from step 4 derived from pGreen_c(G/C/B)FP_GolGat_1F, mix the following in a thermocycler tube

• Run the assembly program in your thermocycler with the lid set to 105 C

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6. Heat shock Golden Gate assembly into chemically competent E coli cells

• Thaw pre-aliquoted 25 ul E coli cells on ice for 30 min

• Add 2 ul of assembly to cells and keep on ice for 15 min

• Heat shock cells at 42C for 45 sec and immediately place on ice

• Mix 500 ul outgrowth media with cells and plate 100 ul

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Resources

• iGEM's Cloning Guide

• IDT Oligo Resuspension Protocol

• IDT Oligo Annealing Protocol

• McClean Lab OpenWetware Golden Gate Protocol

• Addgene's Bacterial Transformation Protocol

• Addgene's LB Agar Plate Prep Protocol

• Microwave LB Agar Plate Prep

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Lab notebook

We received the resuspended oligonucleotides from Noah, our TA for this assignment. First we diluted the oligo pairs in PCR compatible eppendorf tubes, 80 ul of water + 10 ul of each oligo, that way they would annealed and make duplexes.

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Then we prepared the Golden gate annealed duplex fragments that would react as PCR primers mixing each oligo pair dilution (8ul) with T4 DNA ligase Buffer(1ul) and T4 Polynucleotide Kinase (1ul). We left the tubes in the thermocycler and programmed it for 37C incubation for 1 hour, 95C heating for 5 minutes and lastly gradually cool at Room Temperature (~25C) for another hour.

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During this process the T4 PNK catalyzes the transfer and exchange of Pi from the γ position of ATP to the 5 ́ -hydroxyl terminus of polynucleotides. PNK also catalyzes the removal of 3 ́-phosphoryl groups from 3 ́-phosphoryl polynucleotides. T4 PNK requires ATP for activity, but its supplied reaction buffer does not contain ATP because it interferes with radiolabeling reactions. Typically, a kinase reaction is followed by a ligation reaction. To simplify this process, T4 PNK  is optimized for use in T4 DNA Ligase reaction buffer (which contains the appropriate amount of ATP). That way we can then proceed to ligation without a buffer change or heat inactivation.

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Then we prepared the other Golden Gate fragments by mixing the primer pairs (1,25 ul each) with a GFP template (1ul), dNTPs (1ul), Phusion Polymerase Buffer (10ul), Phusion DNA Polymerase (1ul), and water (34,5ul). We ran the PCR reaction following the program below in the thermocycler:

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• Initial denaturation - 1 cycle (98C for 30 seconds)

• PCR - 30 cycles 

      1.Denaturation (98C for 10 seconds)

      2.Annealing (60C for 30 seconds)

      3.Extension (72C for 30 seconds)

• Final extension - 1 cycle (72C for 5 minutes)

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During a typical PCR, cycles of denaturation, annealing and extension are repeated to achieve exponential amplification of the target sequence. Denaturation consists of heating the samples up to a high temperature (typically 94-98°C) to cause denaturation of the template DNA, disrupting the hydrogen bonds and base stacking interactions that hold the DNA strands together. Once the strands are separated, the temperature is decreased to the annealing temperature to allow the primers to base pair (or anneal) to complimentary regions of the template. The annealing temperature is typically between 48-72°C. During the extension step (typically 68-72°C) the polymerase extends the primer to form a nascent DNA strand. This process is repeated multiple times (typically 25-35 cycles). The final step of the PCR is generally a longer, single temperature step (often 5-10 min at 68-72°C) that allows for the completion of any partial copies and the clearance of all replication machinery from the nascent DNA.

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DNA Polymerase helps correct DNA sequences during amplification, lowering the error rate. Once the program finished, we ran the PCR reactions on agarose gel electrophoresis. We cut the gel and then extracted the correct-sized bands with a Gel Extraction Kit from Qiagen (centrifuge in tubes with columns 3 times, the first time with the extraction buffer and the other 2 times with water). The DNA extracted was measured in a Nanodrop in order to confirm the amplification during PCR. 

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After this process we combined and assembled the Golden Gate reactions mixing each fragment derived from pGreen_n(G/B/C)FP_Golgat_1F from the previous step with the primers (1ul each), Cutsmart Buffer (2ul), ATP (2ul), T7 DNA ligase (1ul), BbsI-HF (1ul) and water (8ul). 

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We ran these assemblies in the thermocycler following the program below:

1st Stage: 30 cycles 

• Digest (37C for 5 minutes)

• Ligate (16C for 5 minutes)

2nd Stage: 1 cycle

• Heat denature (55C for 10 minutes)

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Once we finished this step, we proceeded to the bacterial transformation. We thawed 25ul of E. coli competent cells in ice for 30 minutes. We added 2ul of our Golden Gate Assembly to the cells and kept them on ice for 15 more minutes. Then we gave the cell a heat shock of 42C for 45 seconds in a water bath and immediately placed them on ice. We mixed 500ul of outgrowth media with the cells. And finally plated 100ul of each sample in Petri dishes filled with culture medium and antibiotics.