核酸定序設施 DNA Sequencing

樣品準備
Sample preparation /

• 為確保實驗品質，所有樣品務必經過純化去除多餘的dNTP、引子、鹽類及蛋白質再送樣，同時建議以膠體電泳確認DNA產物之單一性。

 

• Plasmid DNA純化後需使用分光光度儀進行OD260/280之測定，一般建議之比值需於1.8上下。

 

• 樣品DNA請以nuclease-free water或DI water回溶，不建議將樣品DNA溶於TE buffer或含有EDTA等抑制物之溶液內。

 

• 樣品請依數量多寡盛裝於0.2ml PCR小管、0.2ml PCR八連排或0.2ml 96孔盤。
   
• 每一個送件管子內需包含: (1)正確濃度的DNA (2)單一股正確濃度引子 (如您選擇五種其中之一的引子由我們來為您添加，則略過這項) (3)補水至體積為10ul。

 

• 樣品取量標準請參考下表

DNA種類	每管建議取量
plasmid/dsDNA	500 ~ 750ng
ssDNA	62.5 ~ 125ng
PCR product	200 ~ 500bp: 7.5 ~ 25ng 500 ~ 1,000bp: 12.5 ~ 50ng 1,000 ~ 2,000bp: 25 ~ 100ng
cosmid/BAC	1.25 ~ 2.5μg
custom primer	0.5 ~ 1μM final concentration
total volumn	10μl

 

• 本設施有提供下列11種常用引子，若樣品已於定序申請單上指定這些引子，則該樣品內不用再加入引子，取好適當的DNA量後直接以DI water補水至10μl即可。​
   

  引子名稱	引子序列
  M13 Forward	TGT AAA ACG ACG GCC AGT
  M13 Reverse	CAG GAA ACA GCT ATG AC
  T7 Promoter	TAA TAC GAC TCA CTA TAG GG
  T7 terminator	GCT AGT TAT TGC TCA GCG G
  SP6 Promoter	AT TTA GGT GAC ACT ATA G
  T3 Promoter	ATT AAC CCT CAC TAA AGG GA
  U6 promoter	TAC AAA ATA CGT GAC GTA G
  CMV forward	CGC AAA TGG GCG GTA GGC GT
  BGH reverse	TAG AAG GCA CAG TCG AGG
  EGFP-C	CAT GGT CCT GCT GGA GTT CGT G
  EGFP-N	CGT CGC CGT CCA GCT CGA CCA G

       

**CMV forward/EGFP-C/EGFP-N/T7 terminator/BGH reverse/U6 promoter are provided since 2021 Feb 24.

  

 

• 樣品標示：

0.2ml PCR小管 - 樣品少於8個適用。請於蓋子頂部或是側邊管壁清楚標示訂單編號及順序(如100-1，100-2....)，並確認與申請單內排序相同。

 

0.2ml PCR八連排 -樣品超過8個以上可使用。 八連排內樣品盛裝順序務必與申請單中排序相同，並須在每條八連排上標明方向順序(至少標註1 和8的位置，9和16...以此類推)，中間不得有任何空位，請連續置放。管上請以彩色膠帶上註明訂單編號和您的姓名，以利作業。如八連排蓋子為單一管子開蓋型的，請將開口朝左邊置放。

 

0.2ml 96孔盤 -樣品超過48個以上可使用。 盤內樣品盛裝順序務必與申請單中排序相同，樣品放置方向為直排開始，A01-H01，A02-H02......A12-H12，8個為一排，中間不能有空位。並於封膜上標示訂單編號。(請勿使用熱壓封膜，避免撕不開)

• Before sequencing, you must remove all residual PCR primers, unincorporated nucleotides, salt and protein from the DNA sample. Furthermore, we strongly recommend check the size and purity by agarose gel electrophoresis.

 

• Plasmid DNA should measure the ratio of the absorbance at 260 and 280 nm after purify. The ratio of sample should be around 1.8.

 

• Please dissolve DNA in nuclease-free water or DI water, dissolving DNA in TE buffer is not recommend.

 

• Please transfer DNA into 0.2ml PCR tube, 8-strip or 96 well microplate, depending on quantity of samples.
• Each tube should contain: (1)DNA (2)One user primer (you may also select one primer which we provide) (3)Add water to 10ul (total volume).

 

• Following is the table of DNA amount used in sequence

Template Type	DNA Amount
plasmid/dsDNA	500 ~ 750ng
ssDNA	62.5 ~ 125ng
PCR product	200 ~ 500bp: 7.5 ~ 25ng 500 ~ 1,000bp: 12.5 ~ 50ng 1,000 ~ 2,000bp: 25 ~ 100ng
cosmid/BAC	1.25 ~ 2.5μg
custom primer	0.5 ~ 1μM final concentration
total volumn	10μl

 

• We offer 11 universal primers in sequencing. If you chose these 11 primers in DNA sequencing-work request form, it is not necessary to add primer to sample anymore. Just transfer appropriate amount of DNA into tube and adjust the volume with DI water to 10μl.
   

  Primer	Sequence
  M13 Forward	TGT AAA ACG ACG GCC AGT
  M13 Reverse	CAG GAA ACA GCT ATG AC
  T7 Promoter	TAA TAC GAC TCA CTA TAG GG
  T7 terminator	GCT AGT TAT TGC TCA GCG G
  SP6 Promoter	AT TTA GGT GAC ACT ATA G
  T3 Promoter	ATT AAC CCT CAC TAA AGG GA
  U6 promoter	TAC AAA ATA CGT GAC GTA G
  CMV forward	CGC AAA TGG GCG GTA GGC GT
  BGH reverse	TAG AAG GCA CAG TCG AGG
  EGFP-C	CAT GGT CCT GCT GGA GTT CGT G
  EGFP-N	CGT CGC CGT CCA GCT CGA CCA G

 

• Single tube: Your samples must be in 0.2 ml flip-top micro-centrifuge tubes, no-stick on labels or tape. Please make sure your sample name should be accord with the application form(label : order no -1, order no -2)

• 8-strip tubes: If you request over 8 samples, please put your samples into the 8-strip tubes, and put them on the 96-well plate rack, label you order number  and your name clearly.

• 96-well plate：If you request over 48 samples, you may put your samples to the 96-well plate , seal with the aluminium foil tape tidely. And label you order number and your name clearly.