DNA sequencing core laboratory is housed in R2-5214, 5F, Research Building II at NHRI. Drop off samples in the boxes or on the racks in the freezer outside the room R2-5211 and a prior web submission form must be submitted with the samples together. NHRI users working off the Zhunan campus, please submit samples by mailing to the Core. The mailing address is:
R2-5214, 5F, 35 Keyan Road,
Zhunan, Miaoli County 35053, Taiwan
(1) After completed the application form online, please print it out and have the principal investigator of laboratory to sign the application form before submitting. Xero copies may not be accepted.
(2) In the online application system, only 10 characters are allowed to name each sample. Avoid using same file name to different samples. The acceptable characters are:
Numbers: 1 2 3 4 5 6 7 8 9 0
Alphabet letters: A B C D E F G H I J K L M N O P Q R S T U V W X Y Z
a b c d e f g h I j k l m n o p q r s t u v w x y z
Symbols: + - # ( )
Please prepare your samples in the following manner: mix DNA template and one set of primer (10 μM / 1 μl) in a single 0.5 ml eppendorf tube in the proportion indicated below. Final sample volumes should be 8 μl. If they are lower, add Milli-Q water to bring them to volume. If user needs the Core to add cost-free primer to the samples, please have the submitted sample volumes in 6.5 μl.
(1) Template and Primer:
Type of Template
PCR Product* 200 – 500 bp
3 – 4 μl
PCR Product* 500 – 1000 bp
5 – 7 μl
Plasmid OD260/OD280 ratio = 1.75 – 1.89
OD260/OD230 ratio = > 2.2
400 – 500 ng
*In the preparation of PCR products, be sure that the staining of the 2 μl PCR product band is stronger than the 500-bp band staining of the 2 μl 100 bp DNA Ladder Marker during the agarose gel electrophoresis.
Sequencing primers provided by the Core Lab at no additional charge:
T7 Promoter Primer
5' - TAA TAC GAC TCA CTA TAG GG - 3'
5' - GAT TTA GGT GAC ACT ATA G - 3'
-21 M13 Forward Primer
5' - TGT AAA ACG ACG GCC AGT - 3'
M13 Reverse Primer
5' - GGA AAC AGC TAT GAC CAT G - 3'
Please indicate on your application form if you would like us to add a primer at the facility.
(2) Sample Labeling:
A. Microfuge tube: on the surface of tube cap, please write on user’s English initial (same as on the application form) and the sample number; on the side of the tube, please label the sample name (use 10 characters only). Be sure that all the labeled information on the tube is the same as the information you provided on the online application form.
B.96-Well plate: Use a 0.2 ml 96-well PCR plate andsealed with aluminum adhesive seals. Please be sure to label the plate.
4.The samples that are already run through the DNA sequencing reactions must be placed on the 96-well plate that is compatible with ABI3730 DNA Analyzer and have gone through the ethanol precipitation. Then submit the samples in a frozen status to the Core within 48 hours after reaction for analyzing.
5.Excess PCR primers and dNTPs must be removed prior to sequencing. During the DNA sequencing, only one set of new primer is allowed to add into the purified DNA template.
6.Ladders provided by the Core for fragment analysis at no additional charge are:
For STR: GS-600Liz and GS-500Liz are provided.
For SNaPshot: GS-120Liz is provided.
If not using the above ladders, please prepare your own ladder to use.
7.To design the primer for SNaPshot use, please follow the length rule, such as 20, 36, 44, 52, 60, 68 …, etc.
8.The raw data of DNA sequencing will only be exported and saved as a computer filename, not text file (.txt). The fragment analysis data will be saved in both the original file and the analyzed text file (.txt).
9.The guideline and policy are made to speed up the whole process of the service and to handle samples more efficiently. If users submit any unclear or unmatched information on the application form or on the sample labeling, the Core reserves the right to reject users’ applications.