【Experimental Log】2015/2/26

2 Sample:CP48 (B. nantoensis), CP51 (B. kawakamii)

DNA extraction: EasyLid (2/26, with 宜軒,總體積為45 ug。)

使用分光光度計 (2+98 uL),測濃度是否足夠。CP48: 0 ug/mL,  260/280,  260/230。CP51: 206.3 ug/mL, 1.27 260/280, 0.69 260/230。(45-2 ug= 43ug, 此兩sample剩下量)

Next: CP48,第一次抽B. nantoensis,就濃度太低,沒有結果。覺得很詭異,因為兩個sample是一起做的。CP51,最後的DNA有一點點顏色 (棕色)。OD260: 測DNA, RNA (核酸);OD280: 測protein;OD230: 測鹽類。OD260/OD280: 1.7~1.9,1.8為佳 (DNA);1.9~2.1,2.0為佳 (RNA),低表protein汙染。OD260/OD230: 2.0~2.4 (DNA);1.8~2.0 (RNA),低表鹽類汙染 (不要低於1.5)。

廣告

【Experimental Log】2015/2/24

5 Sample:a2 (B. aristatoserrulata), a5 (B. aristatoserrulata, not sure), r1 (B. ravenii), CP (B. aristatoserrulata), CP68 (B. kawakamii)

使用分光光度計 (2+98 uL),測濃度是否足夠。a5: 336.5 ug/mL, 1.95 260/280, 2.29 260/230。a2:126.5 ug/mL, 1.78 260/280, 2.57 260/230。r1: 133.5 ug/mL, 2.03 260/280, 1.98 260/230。CP68: 21.9 ug/mL, 1.45 260/280, 0.41 260/230。CP: 13.2 ug/mL, 1.29 260/280, 0.37 260/230。

RAD實驗要求:protocol中,每個樣品起始使用量為 1ug 的gDNA (體積不超過44uL,和Brendan做的時候,最後TE buffer僅加45mL;和宜軒學姊做,他加200mL,故總體積和濃度有差)。每個樣品都準備 3ug 的起始量。計算ex: 舉r1為例,133.5*(43/1000)=5.7405 ug  [133.5是測到的r1的濃度(ug/mL),43是DNA樣品的總量(uL),1uL=0.001mL],5.7405 ug > 3 ug [r1濃度足夠],43 uL,亦沒有超過44*3uL (要處理成三管,故乘三),所以符合實驗所需。舉CP為例,13.2*(200/1000)=2.64 ug2.64 ug3 ug [CP濃度不足夠]。

所有樣本已編成我的編號CP開頭https://docs.google.com/spreadsheets/d/1g78hg1sslijTGtFi5ocrpp1w78b0KeIYEjZqtnKafNY/edit#gid=0 (a5: CP0091, a2: CP0090, CP: CP0082…) 之後就使用Champ Berberis Sample List中的CP開頭編號。

Next: 雖然濃度足夠,但內容物的品質是否夠好?DNA跑膠?還是?希望在3/2之前拿出樣本做。如果可以,想使用r1 (或那系列的B. ravenii) 和CP68 (B. kawakamii),這樣就可以在兩分支中 (Brendan的世界小檗樹,台灣那支系) 在其中各取一個sample,也各取兩種實驗protocol (Brendan和宜軒的方法),拿去測試RAD實驗,哪種合適或較好。上次PCR的a5沒有P出來,但測出的濃度極高,是為什麼?

【Experimental Log】2015/2/22-23

10 Sample:a1 (B. aristatoserrulata), a2 (B. aristatoserrulata), a5 (B. aristatoserrulata, not sure), r1 (B. ravenii), r2 (B. ravenii), r3 (B. ravenii), r4 (B. ravenii), r⑤ (B. ravenii), a① (B. aristatoserrulata, 大鬼湖畔), a② (B. aristatoserrulata, 大鬼湖畔). p.s : 1. the number in circle (ex: ②), that is I marked. 2. These samples are from 大小鬼湖 by Brendan. 3. I should use my serial number.

DNA extraction: EasyLid (2/22, with Brendan). 

Polymease Chain Reaction (PCR): (2/23, with Brendan) 試做自己的一個sample:a5 (Is there DNA inside ?) 其餘的做學長的Sample。結果不佳。a5沒有P出來。

Next: DNA測濃度。(分光光度計) 確定是否有DNA和是否濃度足夠。若加入上次和宜軒一起的兩個sample: CP68 (B. kawakamii) 和 CP (B. aristatoserrulata),總共有12個sample,需從中找出兩個可以上機的樣本,3/2需要使用。

初四一從高雄外婆家回來,便直接回實驗室了。這天 (2/22) 是Brendan的29歲生日。尼泊爾王子的小檗paper出爐了 (Systematics and biogeography of Berberis s.l. inferred from nuclear ITS and chloroplast ndhF gene sequences),Brendan嫌棄得很。Strictly speaking, it’s my first time to do MY experiment, I need more practice.  3/2 (Mon) PM 2:00, 農藝系做實驗

濃度問題:DNA濃度是否足夠?