Collaboration of Archaeologists, Historians and Bioarchaeologists During Removal of Clothing from Korean Mummy of Joseon Dynasty

Abstract

Recently, experts from various research fields made an interdisciplinary collaboration on well-preserved cultural or human remains discovered in fifteenth- to nineteenth-century Joseon tombs of Korea. The academic information acquired under the interdisciplinary collaboration was significant to researchers in Korea because it is entirely original and not to be commonly found in any library resource for historians. This report is the first of full, detailed descriptions about the research on Joseon tomb, by which the vivid glimpse of Joseon people’s lives could be reconstructed, based on the clear archaeological, historical and biological evidences.

Appendices

Appendix I. PCR Amplification in This Study

After quantification was done by NanoDrop^TM ND-1000 Spectrophotometer (Thermo Fisher Scientific, MA, USA), the mixture for PCR amplification of DNA was prepared. 40 ng of DNA was mixed with premix containing 1 mg/ml of BSA (New England Biolabs, MA), 10 pmol of each primer (Integrated DNA Technology, USA), 0.25 mM of dNTP mixture (Invitrogen, USA), 1X PCR buffer, 2 mM of MgSO₄ and 1 unit of Platinum^TM Taq Polymerase High fidelity (Invitrogen, USA). PCR conditions used in this study were as follows: pre-denaturation at 94 °C for 10 min; 38 cycles of denaturation at 94 °C for 20 s; annealing at 56 °C for 10 s; extension at 72 °C for 30 s; final extension at 72 °C for 10 min. PCR amplification was performed using a PTC-200 DNA Engine (Bio-Rad Laboratories, Hercules, CA). Primer sets used for this study were as follows. 288-bp PS I: F15971 (5’-TTA ACT CCA CCA TTA GCA CC-3’) and R16258 (5’-TGG CTT TGG AGT TGC AGT TG-3’); 267-bp PS II: F16144 (5’-TGA CCA CCT GTA GTA CAT AA-3’) and R16410 (5’-GAG GAT GGT GGT CAA GGG AC-3’); 244-bp PS III: F15 (5’-CAC CCT ATT AAC CAC TCA CG-3’) and R258 (5’-GTT ATG ATG TCT GTG TGG AA-3’); 330-bp PS IV: F155 (5’-TAT TTA TCG CAC CTA CGT TC-3’) and R484 (5’-TGA GAT TAG TAG TAT GGG AG-3’) (Holland and Huffine 2011).

Appendix II. Cloning of Amplified DNA Fragment in This Study

When PCR products of 20 μl could be separated as specific bands (288 bp for PS I; 267 bp for PS II; 244 bp for PS III; 330 bp for PS IV) by a 2 % agarose gel electrophoresis, they were purified with QIAqucik Gel Extraction Kit (Qiagen, Germany).

Electrophoresis showed specific bands for PCR products of HD-2 case both in Lab A and B results (288 bp for PS I; 267 bp for PS II; 244 bp for PS III; 330 bp for PS IV). PCR in Lab B was repeated two times. EC, extraction (negative) control.

Ligation of amplified fragment into plasmid vector was done with pGEM-T Easy Vector System (Promega, USA). Briefly, quantity of extracted DNA was measured again by NanoDrop^TM ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). 10 ul of ligation mixture (5 μl of 2X Rapid Ligation buffer, 1 μl of pGEM-T Easy Vector, 1 μl of T4 DNA ligase and 3 μl of gel extracted DNA) was incubated overnight at 4 °C; and after then, the vector was transformed into competent cells (ECOS-101, Yeastern Biotech, Taipei, Taiwan). They were then plated on LB agar plate (50 μg/ml of ampicillin, 0.5 mM IPTG, and 40 ug/μl X-GAL); and were incubated at 37 °C for 12 h. Selected white colonies were incubated in 5 ml of LB Broth, with shaking for 12 h (at 37 °C, 220 rpm). After Plasmid in cultured bacteria was isolated with QIAprep spin miniprep kit (Qiagen, Germany), sequencing was done on each strand using ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, USA) according to manufacturer instruction. The sequence reaction products were analyzed by ABI Prism 3100 automatic sequencer (Applied Biosystems, USA).