Purification of PCR Products and Ligation into T/A plasmid

The gel extraction protocol

1. Cut the DNA band of interest out of the gel using a clean spatula. Place the gel slice to a labeled microfuge tube. IMPORTANT: UV light damages DNA so keep UV light on low intensity and turn the light off right after cutting around the band of interest. <5 seconds total exposure.

2. Estimate the gel slice volume (usually around 100 uL) and add 3 volumes of QG solution.

3. Incubate at 50 degrees C for 10 minutes until gel has dissolved. Give a quick vortex after 5 minutes to help dissolve the gel.

4. Add 1 gel volume of isopropanol to the sample and mix.

For example, if the agarose gel slice was 100 uL, add 100 µL isopropanol. This step increases the yield of DNA fragments <500 bp and >4 kb. For DNA fragments

between 500 bp and 4 kb, addition of isopropanol has no effect on yield and can be omitted.

5. Place a QIAquick spin column in a provided 2 ml collection tube.

6. To bind DNA, apply the sample to the QIAquick column, and centrifuge for 1 min. on highest speed. The maximum volume of the column reservoir is 800 µl. For sample volumes of more

than 800 µl, simply load and spin again.

7. Discard flow-through and place QIAquick column back in the same collection tube.

Collection tubes are re-used to reduce plastic waste.

8. To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 min.

9. Discard the flow-through and centrifuge the QIAquick column for an additional 1 min.

IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless

the flow-through is discarded before this additional centrifugation.

10. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.

11. To elute DNA, add 40 µl of Buffer EB or H₂O to the center of the

QIAquick membrane (do not touch membrane with tip), let the column stand for 1 min, and centrifuge the column for 1 min.

12. Label tube as QF (for Qiagen Fragment), either v (for vector fragment) or i (for insert fragment), the gene or plasmid name, your initials, and date. Also note PCR fragment or digested fragment (example: Bam + EcoRI).

Ligation of purified PCR fragment into pGEM-T Easy vector

T-A Cloning: Taq DNA polymerase and most other polymerases have a terminal transferase activity that results in the non-templated addition of a single dA nucleotide to the 3'-ends of PCR products. This single A “sticky end” can be utilized to clone into a linear plasmid containing single T sticky ends, like pGEM-T Easy.

Ligation reaction set-up

Reagents		·
2X ligation buffer	5 ul
pGEM-T Easy vector (50 ng)	1 ul
Purified DNA fragment (the insert)	3 ul
Ligase enzyme (3 units)	1 ul
Total volume	10 ul

· Gently mix the reactions by pipetting up/down a few times. Spin down if needed.

· Incubate the reactions overnight at 4 degrees C.

· Next, you will transform competent E. coli cells with the DNA ligation reaction.

Ligation time vs number of transformed colonies