APh/BE 162 - DNA Science Lab

This week, the desired DNA was retrieved from the previous electrophoresis agarose gel and purified. To insert our desired DNA fragment into the bacteria plasmids, ligation was first performed to join the complementary sticky ends (that were cut by the restriction enzymes last week) onto the vectors. Subsequently, cells containing DH5a strains were transformed to allow the bacteria plasmids to take up the modified vectors.

1. Desired DNA bands on the gel are cut out and isolated in eppendorf tubes. The weight of the gel is noted to be about 1.61g.

2. Isolated gel is dissolved in Buffer QG from the Qiagen Gel Extraction Kit. Amount of buffer to be added is 3 times the weight of the gel (i.e. 4.83 ml). Dissolution is allowed to occur at 50degC for about 5-10 minutes.

3. Proceeding gel extraction steps were carried out as per the Qiagen Gel Extraction Assay. Briefly, isopropanol is added to increase the yield of the desired DNA fragments. Subsequently, it is then centrifuged @ 13,200g for 1 min to retain the DNA in the column. This process is repeated until the entire volume is depleted. To further remove the agarose gel, Buffer PE is added and centrifugation is repeated.

4. To elute the DNA, Buffer EB is added and again centrifuged. The final DNA was collected in the filtrate.

1. HindIII and KpnI were added to the previous PCR products to obtain “sticky ends”.

Briefly, 5 volumes of Buffer PB is added to the PCR fragments and centrifuged for 1 min to bind the DNA to the column. Buffer PE is added to wash the PCR extracted, and again centrifuged for 1 min @ 13,200g. To elute the desired PCR product, buffer EB is added, centrifuged and the final PCR product is collected in the filtrate.

Briefly, mixture of various proportions* of vector and insert were added to conc. DNA Dilution Buffer to a final concentration of 10µL. 10µL of T4 DNA ligation buffer is then added and mixed thoroughly. Finally 1 µL of T4 DNA ligase is added and the mixture is incubated at 15-25degC for 5 mins.

Ratio	Vector (µL)	Insert (µL)	DNA dilute buffer (µL)
1:3	3.6	2.8	3.6
1:1	1.2	2.8	6.0
3:1	0.4	2.8	6.8
No insert	1.2	0	8.8
No vector	0	2.8	7.2

2. The same plasmid was treated with a killer cut to ligate the center of the LacZ DNA sequence to prevent religation and so that cells containing this killer cut do not produce and eventually die. This serves as a control for each ratio of vector and insert.

1. Electrocompetent E. Coli (Escherichia coli) cells were grown to an OD of 0.6, washed with glycerol and allowed to incubate in LB for 30 mins before electroporation. This is to allow the cells to develop some anti-biotic resistance and not lose their plasmids later during transformation.

2. Electroporation is the process where cells are allowed to uptake a foreign DNA by means of stressing the cells via an electric field. This process was carried out as per the same Roche Rapid DNA Ligation Kit protocol. Briefly, 100 µL of Binding Buffer is added to the ligation reaction mixture obtained in step 1 and centrifuged. 500 µL of wash buffer is then added to the upper reservoir of the filter tube and again centrifuged. This process is repeated twice. 100 µL of sterile double distilled water is finally added, centrifuged and the flowthrough is retained. Roughly 10 µL of the flowthrough is then added to the electrocompetent cells in Step 1 and then subjected to electroporation. The settings for electroporation were: 2.5 kV, 25 MF, 200ohm.

3. The electroporated cells were then plated and grown @ 37degC for 12-15 hours. The following were added in the agar where cells were grown:

Shown below, was the Petri dish containing no X-gal. As expected, no blue color can be seen.

In the other type of cells containing the strain DH5a-Z1/MC4100Z1, the TEC repressor is present, and hence only with the addition of ATC inducer, can Lac Z be expressed. Hence as shown in the dish on the left (ATC absent), virtually no blue color could be detected. This is compared to the vivid blue color found in the dish on the right, with the presence of ATC.

Also plated were the pZE21-GFP cells (for fun) – fluorescence was observed as expected.