New ligation/transformation for Bn PUM1 F2, Mm MAEL F2, Mm DDX4 R2, Mm PIWIL1 R1, Mm PUM, Mm TDRD1

08/01/12

Set a new ligation for Bn PUM1 F2, Mm MAEL F2, Mm DDX4 R2, Mm PIWIL1 R1, Mm PUM, Mm TDRD1; + 2 control tubes using the same amount (1.5 µl) of the control DNA insert.

09/01/12

Transformation using 3 µl of ligation per tube, except for Control 2 where I added 5 µl of ligation. Plated with 400 µl and waited plates to be completely dry to incubate at 37 °C at 19:00.

NanoDrop

Chema suggested to quantify the amount of DNA in the PCR products to be able to calculate the quantity to be used in the ligation reaction. To do this I used NanoDrop spectophotometer.

Bn PUM1 F2 Mm MAEL F2 Mm DDX4 R2 Mm PIWIL1 R1 Mm PUM Mm TDRD1
260/280 2.54 2.02 1.87 0.63 2.12 2.21
260/230 0.77 1.47 0.29 -0.02 0.79 0.41
ng/µl 56.3 207.7 24.0 -6.2 62.6 64.3

260/280: ratio of sample absorbance at 260 and 280 nm. The ratio of absorbance at 260 and 280 nm is used to assess the purity of DNA and RNA. A ratio of ~1.8 is generally accepted as “pure” for DNA; a ratio of ~2.0 is generally accepted as “pure” for RNA. If the ratio is appreciably lower in either case, it may indicate the presence of protein, phenol or other contaminants that absorb strongly at or near 280 nm.

260/230: ratio of sample absorbance at 260 and 230 nm. This is a secondary measure of nucleic acid purity. The 260/230 values for “pure” nucleic acid are often higher than the respective 260/280 values. They are commonly in the range of 1.8-2.2. If the ratio is appreciably lower, this may indicate the presence of co-purified contaminants.

ng/uL: sample concentration in ng/uL based on absorbance at 260 nm and the selected analysis constant.

From NanoDrop User Guide.

According to Aina, the contamination at the 260/230 is from the extraction buffer and should not interfere with the ligation.

10/01/12

Colony growth was better this time, but I still should be getting more colonies than now for some samples. Increase in colony number might be due to the plating amount (400 µl) or even the 1 µL increase in the PCR product for the ligation.

Gene Colonies Picked
Bn PUM1 F2 ~10 6
Mm MAEL F2 3 3
Mm DDX4 R2 Many 6
Mm Piwil1 R1 5 5
Mm PUM ~15 6
Mm TDRD1 ~10 6
Control 1 7 5
Control 2 Many 5

Colony checking PCR started at 14:00.

11/01/12

No positive colonies… Repeat.

Ms_Colony2_110112MmColony 2012-01-11 12hr 23min