Spider DNA degrade quickly? / DNA labah-labah rosak cepat?

Situation:
The spider collected from the field were stored in 75% ethanol. The specimens were left in the chalet for about a month in the forest. As soon as we got back to our lab, the spider specimen were stored in temperature -20oC freezer. After 18 months, we tried to extract the DNA and amplify at the COI (600-700 bp) and 18S (900-1100 bp) region, we found out that most DNA had been degraded. This is not surprising as previous report also mentioned that spider DNA degrade very quickly in less than 2 months if stored in 75% ethanol.

Recommendation: PLEASE extract the DNA as soon as possible.


Situasi:
Labah-labah yang dikumpul dari lapangan telah disimpan dalam 75% etanol. Spesimen dibiarkan di chalet selama kira-kira sebulan di dalam hutan. Sebaik sahaja kami kembali ke makmal, spesimen labah-labah telah disimpan di dalam peti sejuk pada suhu-20oC. Selepas 18 bulan, kami cuba untuk mengekstrak DNA dan menguatkan menggunakan COI (600-700 bp) dan 18S (900-1100 bp), namun kita mendapati bahawa kebanyakan DNA telah rosak. Ini tidak menghairankan kerana laporan sebelum ini juga menyebut bahawa labah-labah DNA rosak dengan cepat dalam masa kurang dari 2-6 bulan jika disimpan dalam 75% etanol.

Nasihat: SILA mengekstrak DNA spesimen secepat mungkin.

Polymerase Chain Reaction (PCR)

The PCR mixture can be divided into several mixture:

1. Using PCR mastermix (1 sample = 50 microliter)

a. water (dH2O)      = 10 microliter
b. Primer forward     = 5 microliter
c. Primer reverse      = 5 microliter
d. Mastermix            = 25 microliter
e. DNA sample        =  5 microliter


2. Using manual PCR mixture (1 sample = 25 microliter)

a. Buffer polymerase (10x)            = 5.5 microliter
b. Magnesium Chloride (25mM)    = 2.5 microliter
c. dNTPs                                      = 1 microliter
d. Primer forward                          =  2.5 microliter
e. Primer reverse                           =  2.5 microliter
f. Taq polymerase                          =  0.2 microliter
g. water dH2O                              =  5.8 microliter
h. DNA sample                              = 5 microliter

Add-in BSA if MUST to enhance PCR product if the DNA quality is poor.