Plasmid vector being used : pNZ8148

Intermediate steps repeated for 35 cycles

Mix:

• Template solution - 10uL
• FWD primer - 2.5uL
• Rev primer - 2.5uL
• HF buffer - 10uL
• dNTPs - 1uL
• Polymerase - 0.5uL
• dH2O - 23.5 uL

Total - 50uL

• Fragment + dH20 -> 16uL
• Bsa1-HF -> 0.5uL
• T4 Ligase -> 0.5uL
• T4 Ligase Buffer - 2uL

Run :

• 37C -> 5min
• 37C -> 5min
• 16C -> 5min
• Repeat above 2 steps for 30 cycles
• 60C -> 15min

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
2. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg gel ~100 μl). Note: For >2% agarose gels, add 6 volumes of Buffer QG.
3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). Vortex the tube every 2–3 min to help dissolve the gel. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 μl of 3 M sodium acetate, pH 5.0, and mix. The mixture turns yellow.
4. Add 1 gel volume of isopropanol to the sample and mix.
5. Place a QIAquick spin column in a provided 2 ml collection tube or into a vacuum manifold. To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min or apply vacuum to the manifold until all the samples have passed through the column. Discard flow-through and place the QIAquick column back into the same tube. For sample volumes >800 μl, load and spin/apply vacuum again.
6. If DNA will subsequently be used for sequencing, in vitro transcription, or microinjection, add 500 μl of Buffer QG to the QIAquick column and centrifuge for 1 min or apply vacuum. Discard flow-through and place the QIAquick column back into the same tube.
7. To wash, add 750 μl of Buffer PE to the QIAquick column and centrifuge for 1 min or apply vacuum. Discard flow-through and place the QIAquick column back into the same tube.
8. Place the QIAquick column into a clean 1.5 ml microcentrifuge tube.
9. To elute DNA, add 50 μl of Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30 μl of Buffer EB to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min. After the addition of Buffer EB to the QIAquick membrane, increasing the incubation time to up to 4 min can increase the yield of purified DNA.

1. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube. Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet. If LyseBlue reagent has been added to Buffer P1, vigorously shake the buffer bottle before use to ensure LyseBlue particles are completely dissolved. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.
2. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times. Mix gently by inverting the tube. Do not vortex, because this will result in shearing of genomic DNA and contamination of plasmid. Continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min. If LyseBlue has been added to Buffer P1, the cell suspension will turn blue after addition of Buffer P2. Mixing should result in a homogeneously colored suspension.
3. Add 350 μl Buffer N3. Mix immediately and thoroughly by inverting the tube 4–6 times. To avoid localized precipitation, mix the solution thoroughly, immediately after addition of Buffer N3. Large culture volumes (e.g., ≥5 ml) may require inverting up to 10 times. The solution should become cloudy. If LyseBlue reagent has been used, the suspension should be mixed until all trace of blue is gone and the suspension is colorless.
4. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge. A compact white pellet will form.
5. Apply 800 μl of the supernatant from step 4 to the QIAprep 2.0 Spin Column by pipetting.
6. Centrifuge for 30–60 s. Discard the flow through.
7. Recommended: Wash the QIAprep 2.0 Spin Column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow through. This step is necessary to remove trace nuclease activity when using endA+ strains, such as the JM series, HB101 and its derivatives, or any wild-type strain, which have high levels of nuclease activity or high carbohydrate content. Host strains, such as XL-1 Blue and DH5α, do not require this additional wash step.
8. Wash QIAprep 2.0 Spin Column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s.
9. Discard the flow through, and centrifuge at full speed for an additional 1 min to remove residual wash buffer. Important: Residual wash buffer will not be completely removed unless the flow through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions.
10. Place the QIAprep 2.0 Spin Column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep 2.0 Spin Column, let stand for 1 min, and centrifuge for 1 min.