【Experimental Log】2016/02/22-23

16 Samples:CPL001 (B. kawakamii), CPL002 (B. nantoensis), CPL003 (B. nantoensis), CPL004 (B. mingetsensis), CPL005 (B. mingetsensis), CPL006 (B. breviesepala), CPL007 (B. tarokoensis), CPL008 (B. tarokoensis), CPL009 (B. tarokoensis), CPL013 (B. pengii), CPL014 (B. pengii), CPL015 (B. aristatoserrulata), CPL016 (B. aristatoserrulata), CPL017 (B. aristatoserrulata), CPL018 (B. breviesepala), CP227 (B. alpicola)

DNA extraction:CTab (未秤重,目視約一片正常大小葉子,加入液態氮磨碎。) 總體積用50 ug去溶。

純化kit:Monarch™ PCR & DNA Cleanup Kit (5 μg) (NEB)

DNA定量:Qbit

Qbit結果:CPL001 (B. kawakamii):too high ng/uL, CPL002 (B. nantoensis):70.2 ng/uL, CPL003 (B. nantoensis):80.0 ng/uL, CPL004 (B. mingetsensis):112 ng/uL, CPL005 (B. mingetsensis):106 ng/uL, CPL006 (B. breviesepala):too high ng/uL, CPL007 (B. tarokoensis):104 ng/uL, CPL008 (B. tarokoensis):99.4 ng/uL, CPL009 (B. tarokoensis):120 ng/uL, CPL013 (B. pengii):112 ng/uL, CPL014 (B. pengii):76.8(76.4) ng/uL, CPL015 (B. aristatoserrulata):too high ng/uL, CPL016 (B. aristatoserrulata):100 ng/uL, CPL017 (B. aristatoserrulata):98.4 ng/uL, CPL018 (B. breviesepala):91.4 ng/uL, CP227 (B. alpicola):85.0 ng/uL

結論:這次抽活體們。CPL1, CPL5, CPL7 實驗過程中有不小心掉落地面 (似乎沒有很大影響)

【Experimental Log】2016/02/19-20

22 Samples:CP172 (B. kawakamii), CP174 (B. hayatana), CP184 (B. nantoensis), CP169 (B. alpicola), CP194 (B. tarokoensis), CP196 (B. schaaliae), CP197 (B. schaaliae), CP200 (B. aristatoserrulata), CP201 (B. aristatoserrulata), CP209 (B. kawakamii), CP210 (B. kawakamii), CP224 (B. kawakamii), CP225 (B. alpicola), CP226 (B. alpicola), CP227 (B. alpicola), CP233 (B. pengii), CP234 (B. pengii), CP235 (B. pengii), CP236 (B. pengii), CP237 (B. kawakamii)

DNA extraction:CTab (未秤重,目視約一片正常大小葉子,加入液態氮磨碎。) 總體積用50 ug去溶。[新來的!CP233, CP234, CP235, CP236, CP237,因為量多,就都各抽兩支 (-1和-2)。]

純化kit:Monarch™ PCR & DNA Cleanup Kit (5 μg) (NEB)

DNA定量:Qbit

20160219 Qbit結果:CP172 (B. kawakamii):39.8 ng/uL,, CP174 (B. hayatana):7.46 ng/uL, CP184-1 (B. nantoensis):20(20.2) ng/uL, CP184-2 (B. nantoensis):25 ng/uL, CP169 (B. alpicola):46.6 ng/uL, CP194 (B. tarokoensis):45 ng/uL, CP196 (B. schaaliae):42.2 ng/uL, CP197 (B. schaaliae):52.2 ng/uL, CP200 (B. aristatoserrulata):36 ng/uL, CP201 (B. aristatoserrulata):36.2 ng/uL, CP209 (B. kawakamii):51.2 ng/uL, CP210 (B. kawakamii):22 ng/uL, CP224 (B. kawakamii):91.8(92) ng/uL, CP225 (B. alpicola):35.6 ng/uL, CP226 (B. alpicola):0.142 ng/uL, CP227 (B. alpicola):29 ng/uL

20160220 Qbit結果:CP233-1 (B. pengii):too hight ng/uL, CP233-2 (B. pengii):110(112) ng/uL, CP234-1 (B. pengii):too hight ng/uL, CP234-2 (B. pengii):too hight ng/uL, CP235-1 (B. pengii):too hight ng/uL, CP235-2 (B. pengii):too hight ng/uL, CP236-1 (B. pengii):too hight ng/uL, CP236-2 (B. pengii):too hight ng/uL, CP237-1 (B. kawakamii):too hight ng/uL, CP237-2 (B. kawakamii):too hight ng/uL

CP172 (B. kawakamii):29 ng/uL, CP184 (B. nantoensis):45.2 ng/uL, CP224 (B. kawakamii):too hight ng/uL [這支CP172是突然找到,不知從何來的,突然出現的髒原液,沒有日期,和20160219那支CP172(原抽取時間為20151027) 的那管不同;CP148是CP148-1和CP148-2的濃縮成一管;CP224 20160219已測過,20160220重測]

結論:大家都很乖巧,表現也都不錯。只是20160219測得似乎大家都偏低 (怪)。又讓我很意外的是B. alpicola意外的難抽 (抽樣重測幾支,還算準確)。目前冰箱待過cleanup kit的sample都已經結束。下禮拜把活體抽抽完。

【Experimental Log】2016/02/17

6 Sample:CP188 (B. tarokoensis), CP189 (B. tarokoensis), CP190 (B. tarokoensis), CP191 (B. breviesepala), CP192 (B. tarokoensis), CP193 (B. tarokoensis) [還有CP194採集紀錄皆為同一批,酖因為當時想說湊不到雙數,就先暫緩CP194。]

純化kit:Monarch™ PCR & DNA Cleanup Kit (5 μg) (NEB)

DNA定量:Qbit

Qbit結果:CP188 (B. tarokoensis):80.6 ng/uL, CP189 (B. tarokoensis):68.6 ng/uL, CP190 (B. tarokoensis):60.6 ng/uL, CP191 (B. breviesepala):85.8 ng/uL, CP192 (B. tarokoensis):83 (83.2) ng/uL, CP193 (B. tarokoensis):80.6 ng/uL

這批為嵐山鐵道(20151118)那次的採集。當時就已經先抽好了,到屆時才過cleanup kit+Qbit定量。

【Experimental Log】2016/02/17-18

17 Sample:CP210 (B. kawakamii), CP211 (B. kawakamii), CP212 (B. kawakamii), CP213 (B. kawakamii), CP214 (B. kawakamii), CP215 (B. kawakamii), CP216 (B. kawakamii), CP217 (B. kawakamii), CP218 (B. hayatana), CP219 (B. hayatana), CP220 (B. hayatana), CP221 (B. hayatana), CP222 (B. hayatana), CP223 (B. hayatana), CP230 (疑似B. pengii), CP231 (疑似B. pengii), CP232 (疑似B. pengii)

DNA extraction:CTab (未秤重,目視約一片正常大小葉子,加入液態氮磨碎。) 總體積用50 ug去溶。[CP230, CP231, CP232]

純化kit:Monarch™ PCR & DNA Cleanup Kit (5 μg) (NEB) [ALL]

DNA定量:Qbit

Qbit結果CP210 (B. kawakamii):100 ng/uL, CP211 (B. kawakamii):106 ng/uL, CP212 (B. kawakamii):100 ng/uL, CP213 (B. kawakamii):96.6 ng/uL, CP214 (B. kawakamii):99.2 ng/uL, CP215 (B. kawakamii):85.2 ng/uL, CP216 (B. kawakamii):79.8 ng/uL, CP217 (B. kawakamii):95.2 ng/uL, CP218 (B. hayatana):23.2 ng/uL, CP219 (B. hayatana):42.0 ng/uL, CP220 (B. hayatana):50.4 ng/uL, CP221 (B. hayatana):61.0 ng/uL, CP222 (B. hayatana):41.4 ng/uL, CP223 (B. hayatana):51.6 ng/uL, CP230-1 (疑似B. pengii):70 (68.8)ng/uL, CP230-2 (疑似B. pengii):80.2 (79.8) ng/uL, CP231-1 (疑似B. pengii):104 ng/uL, CP231-2 (疑似B. pengii):96.8 ng/uL, CP232-1 (疑似B. pengii):110 (108) ng/uL, CP232-2 (疑似B. pengii):91 ng/uL

結論:大致上狀況都還不錯,都在預期內。

CP230, CP231, CP232是新來的。登山社學長楊斯顯從台灣東南部美奈田山帶回來的。正好是我很缺少的取樣點 (連油脂都沒去過耶,他只聽過那裏有小檗,但不知道是誰)。拿到的後有很多份,但枯得差不多了,但其中有花有果的狀況竟然算好。清楚看到花的最外一輪花萼為尖形,果為圓,加上地理位置,學長推斷他為南台灣小檗 (B. pengii)。但我對南台灣小檗實在不熟悉,無法一時間認出來。利馬用液態氮磨碎抽DNA。
CP210~217的Sample,阿倫在20151207幫我在大雪山採,以及老師在20151208在塔塔加幫我採的台灣小檗 (B. kawakamii)。那時候剛拿到就已經抽好了,一直沒有過colum和測濃度,就剛好用這組新的純化Kit,也剛好有Qbit,就一起做完。
CP218~223的Sample,是在20151214和家人一起去太平山採回來的。一樣當時已經抽好,到屆時才純化+測濃度。
昨天(20160217)翊萱幫我從向陽帶小檗回來。和學長討論後感覺應該是南台灣小檗 (B. pengii)。我沒有辨識成功。對他的多樣化似乎還沒有抓到精隨。明天(20160219)買到液態氮再一口氣做好DNA吧。
冰箱裡還有幾支落單的DNA,找時間也過純化kit+測濃度後再收進盒裡。還有陽台上的活體小檗,也大概要收DNA了。這些事情希望能在下禮拜二,油脂學長回來之前完成。(還要討論出國採集一事,忙碌的一學期要開始了)

加油。

【Experimental Log】2016/02/16

11 Sample:CP162 (B. tarokoensis), CP163 (B. tarokoensis), CP164 (B. tarokoensis), CP164n (B. tarokoensis), CP165 (B. tarokoensis), CP166 (B. tarokoensis), CP167 (B. tarokoensis), CP168 (B. tarokoensis) [8/10抽的藍字蓋那批 (有兩組,另一組為黑字蓋)], CP185 (B. tarokoensis), CP186 (B. tarokoensis), CP187 (B. tarokoensis)

純化kit:Monarch™ PCR & DNA Cleanup Kit (5 μg) (NEB) (之前的Monarch DNA Gel extraction kit,是用在切膠上,這是直接拿來清DNA使用。)

DNA定量:Qbit


 

Monarch™ PCR & DNA Cleanup Kit (5 μg) (NEB)

原始:CP162 (B. tarokoensis), CP163 (B. tarokoensis), CP164 (B. tarokoensis), CP164n (B. tarokoensis), CP165 (B. tarokoensis), CP166 (B. tarokoensis), CP167 (B. tarokoensis), CP168 (B. tarokoensis),用nanodrop測的時候,品質就已經很糟了,依然留有視覺髒色,也沒有濃度 (詳見:https://champ1115.wordpress.com/2015/08/14/%E3%80%90experimental-log%E3%80%91201587-9/)。

過程:全使用Cleanup Kit,依造protocol。

結果:全無。原因可能是本來在過Colum就把DNA通通洗光了。


Monarch™ PCR & DNA Cleanup Kit (5 μg) (NEB)

原始:CP185 (B. tarokoensis):Too high, CP186 (B. tarokoensis):4.78 (5.04) ng/uL, CP187 (B. tarokoensis):87.6 ng/uL

過程:全使用Cleanup Kit,微調protocol,只洗一次。

結果:CP185 (B. tarokoensis):84.6 ng/uL, CP186 (B. tarokoensis):0.18 ng/uL, CP187 (B. tarokoensis):64 ng/uL

結論:

經前後比較測試可發現,最多會掉約20 ng/uL。但也有可能為Qbit測濃度的誤差。但重點是:1.原來濃度足夠使用上機的,Cleanup完後,依然可以。2.髒污或視覺髒色消失。

足矣。

【Experimental Log】2016/02/03-04

5 Sample:760(B. schaaliae), 226(B. alpicola), 227(B. alpicola), 228-2(B. barandana), 229-2(B. barandana), 229-1(B. barandana), 229-1(B. barandana), 107-5 (構樹)

比較純化不同kit: Monarch DNA Gel extraction kit (NEB), T1020S, AJ innuprep DoublePure kit, Wizard® DNA Clean-Up System (Promaga)

DNA定量:Qbit


 

20160203 Promaga vs Monarch

原始:226:100 ng/uL, 227:52.0 ng/uL, 228-2:x, 229-2:0.106 ng/uL (228-2, 229-2為B. barandana,來自英國熊爺爺,但保存很糟糕,抽完DNA後顏色就很重。就先使用Promaga純化,DNA回溶後,視覺上顏色已經清澈。但在Qbit定量後發現幾乎沒有濃度。我將得到的兩個B. barandana的Sample各抽成兩管,故分成228-1, 229-1 (CTAB後的"髒"源液) 和228-2, 229-2 。)

過程:226使用Promaga (想比較Promaga在其他高濃度DNA的樣本表現, vs 228-2和229-2),227, 228-1, 229-1使用Monarch (比較使用前後DNA濃度,以及同個體在不同純化kit後留下的DNA量的差異。)。

結果:226過Promaga後剩下0.36 ng/uL (原來為100 ng/uL)。227過Monarch後為57.2 ng/uL (原來為52.0 ng/uL)。228-1過Monarch後為6.2 ng/uL (過Promaga的228-2無濃度)。229-1過Monarch後為15.1 ng/uL (過Promaga的229-2為0.106 ng/uL)。

結論:

  1. Promaga的純化後效果很差,幾乎也把DNA濃度洗掉。
  2. Monarch的純化效果好。很髒的原液過kit後,還能留下DNA濃度,亦能將視覺髒色洗淨,明顯優於過去kit (過去髒色洗不掉,如之前過的Colum;或者Promaga,留不住DNA)。

 

20160204 AJ測試

原始:107-5 (構樹):too high, 第二次稀釋十倍:25.4 ng/uL (nanodrop:1942.8 ug/mL, 1.81 260/280, 1.35 260/230。CTAB後的原液,視覺上有懸浮物。), 760:104 ng/uL。

過程:使用 AJ innuprep DoublePure kit

結果:107-5 (構樹):too high (靠杯忘記稀釋就又測Qbit,但視覺上已清澈。), 760:93ng/uL (原始為104 ng/uL)。

結果2 (nanodrop):107-5 (構樹):325.5 ug/mL, 1.74 260/280, 1.85 260/230;760:61.4 ug/mL, 1.9 260/280, 1.69 260/230;228-1和229-1:沒測到濃度。

結論:AJ innuprep DoublePure kit效果依然不錯。依然對nanodrop沒什麼好感。