Proceedings of MatricesFor IITTEP – ICoMaNSEd 2015
ISBN: 978-602-74204-0-3
Biology Page 377
2. Materials and Methods
The tools used in this study include: bottle samples, loops, silkworm breeding box test and toxicity test, stereomicroscope Carl Zeis with Camera, 3D hirox Microscope KH-8700,
QiaExel, master cycler eppendorf, tips, eppendorf vial, centrifuse eppendorf, glass objects, laminar air flow, autoclave, centrifuge, heater, analytical balance, etc. petri dish. Materials
used include alcohol 70, nutrient media broth Merck, media nutrient agar Oxoid, media dextro potato agar Merck, peptone, kit DNeasy Blood Tissue Kit QIAGEN, label, leaf
cabbage, C. binotalis, P. xylostella etc.
2.1. Isolation of Bacteria from Caterpillar.
Entomopathogenic bacteria isolation was performed using smear technique Worang and Mokosuli, 2010. Caterpillar body part injured NA applied directly to the media nutrient
agar. Media Inkubasikan for 2 x 24 hours at room temperature. Most Bacterial isolates were grown further in the analysis of the characteristics of isolates among other forms of growth
isolates, form colonies, the ability to reduce glucose and gram staining. Other isolates using a sub-culture in nutrient broth media were prepared for analysis of DNA 16S RNA gene. Most
bacterial culture in nutrient broth media prepared for pathogenicity in vitro test on larvae in cabbage.
2.2. DNA Extraction and Amplification of 16S RNA Gene
DNA extraction from pure cultures of bacteria derived bacterial isolates from caterpillar pests in cabbage in Nutrient Broth media. A total of 3 ml bacterial culture in log phase are taken
and stored in vials of 1.5 ml eppendorf then extracted with protocol according to DNA kit innuPREP DNA Micro Kit, Germany with modifications. DNA is extracted and analyzed
purity and konsenterasinya use nanospektrofotometer. The analysis showed the purity of the extracted DNA was 1.8 at a concentration of 65 ug ml It shows the process of DNA
extraction optimum running so that it can proceed at this stage with metdoe PCR amplification.
DNA sample amplification reaction performed in 0.2 ml PCR tubes. At each PCR reaction tube was added RBC Taq 5 units ml as much as 0:25 mL, 10 x Taq buffer containing Mg
2+ by
5 mL,
4 mL
dNTP 2.5mm
total, universal
primers 63F
5- CAGGCCTAACACATGCAAGTC-3
and 1387R
universal primer
5- GGGCGGWGTGTACAAGGC-3 of each 1:25 mL 20 pmol and 1:25 mL 20 pmol,
extract the genome as much as 2.5 mL 100 ng and plus ddH
2
O until the volume becomes 50 mL. PCR products were taken and stored at 4 ° C for further examined using electrophoresis
qiaexel.
3. Result and Discussion 3.1. Isolation of Entomopathogenic Bacteria
