Webboard : dna extraction

 
Date Content
pie
05-08-2010 21:41:48

During DNA extraction, how 70% EtOH can remove excess salts and other unwanted stuff from DNA solution while before this step absolute alcohol can precipitate DNA?


 


I mean same solution only different in concentration but why it functions not similar???


 


 


Respect to Professors,


Thank you for answer my question!

tc
06-08-2010 01:15:53
the amount of water is the key.

Absolute ethanol forces DNA to precipitate. 70% EthOH has enough water to remove salt and at the same time with enough ethanol to keep DNA as pellet.
pie 1st yr
06-08-2010 21:15:27

Thank you for very clear answer which help me a lot !!!!!!!!! 


Could you explain more about the mechanism of absolute ethanol which can precipitate DNA?


 


i think it associate with the dielectric constant, is that right? Could you give me more details?


 


 


And another question in cloning step, how R-DNA can re-anneal by sodium citrate?


Because before this step R-DNA was denatured by NaOH because of pH but sodium citrate is a kind of salt. Is it can neutralized pH?


 


 


Thank you for your kindness

tc
07-08-2010 23:34:44
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pie
08-08-2010 20:08:48
ไม่ใช่ค่ะ คือหนูพยายามทำความเข้าใจด้วยตัวเองอยู่ แต่ก็ยังงงๆ ค่ะ
tc
09-08-2010 00:51:56
ขอบคุณครับ  ขออภัยที่ให้คอยครับ  เผอิญคำถามของคุณ
thoughtful มาก ผมก็เลยสงสัยว่าเป็นข้อสอบหรือการบ้านรึเปล่า 
พอรู้ว่าไม่ใช่ก็มีความยินดีครับ
เพราะแปลว่านักศึกษามีความตั้งใจในการเรียน



Back to your questions! The first thing to keep in mind is DNA is
hydrophilic especially at its phosphate backbone part. It is prone to
electrostatically interact with water molecules. Of course, this makes
DNA highly soluble!!!



Now, with ethanol, we have a different story. Like you said, it is about
dielectric constant. Ethanol is not as polar as water. In addition,
some ions in the solution can interact with DNA more, and these
interactions neutralize the charges of nucleic acid. Hence, DNA tends to
precipitate.



You might already know that sometimes sodium acetate is added to help
precipitating DNA. Unlike water, ethanol could promote the interaction
between Na+ and the phosphate group, which significantly neutralize the
overall charge.



ผมพยายามใช้ภาษาง่าย ๆ อธิบายครับ ถ้าอยากให้ลึกลงพวก Physical Chem
กว่านี้ ก็บอกได้นะครับ  ผมไม่แน่ใจว่าคุณเป็นปีหนึ่งของอะไร 
ถ้าอยู่ศาลายาก็ถามผ่านบอร์ดได้ครับ ถ้าอยู่พญาไทก็มาถกกันเพิ่มได้ครับ
ธนรรถ
09-08-2010 00:56:50
คำถามที่สองเรื่อง R-DNA กับ sodium citrate กรุณาให้รายละเอียดเพิ่มได้ไหมครับ 

ขอบคุณครับ

nchp
09-08-2010 22:19:37

หนูเรียนปี1 ป.โท ภาควิชาชีวเคมี ศิริราช ค่ะ


 


 


หนูชอบคณะวิทย์ที่มหิดลมากๆๆ ค่ะ


ไม่เข้าใจอะไร ก็สามารถเข้ามาถามได้


 


 


อาจารย์ที่คณะวิทย์ก็เก่งๆ ทั้งนั้น


 


ขอบคุณสำหรับคำตอบนะค่ะ ช่วยให้เข้าใจแบบชัดเจนสุดๆๆ


คือที่อาจารย์อธิบายมา หนูก็เข้าใจแล้วนะค่ะ แต่ถ้าจะเพิ่มลึกลงก็ได้ค่ะ เพราะคงเป็นความรู้ที่หาเรียนในห้องเรียนได้ยาก


 


สำหรับคำถามข้อที่2


ก็คือว่าหนูทำแล็ปเรื่อง cloning ค่ะ แล้วในขั้นตอนของ minipreparation


ที่ตอนแรก centrifuge แล้วเอา supernatant ออก


แล้วเติม solution I,II,III  


โดย solution II ค่ะมีส่วนผสมของ SDS และ  NaOH เพื่อจะ lyse cell และ denatured DNA ตามลำดับ


 แล้วพอเติม solutionIII ซึ่งมี sodium acetate เป็นส่วนผสมค่ะ


 


ซึ่งที่หนูงงก็คือ ทำไม sodium acetate จึงสามารถทำให้ DNA กลับมา reanneal ได้อีกครั้ง ในเมื่อมันเป็นเกลือ


มันจะสามารถช่วยลด pH จากการเติม NaOH ในขั้นตอนก่อนหน้านี้ได้เหรอค่ะ


 


 หรือว่า sodium acetate ไม่เกี่ยวข้องกับการ reanneal แต่เพื่อช่วยในขั้นต่อไปเท่านั้น (ซึ่งเติม absolute ethanol เพื่อให้ตกตะกอน) แต่ถ้าเป็นอย่างนั้นจะ reanneal ตอนไหน หรือว่า final product  จะเป็น single strand ซึ่งมันก็ควรเป็น double strand ไม่ใช่เหรอค่ะ


 


หรือว่าเข้าใจผิดตั้งแต่ต้น 55


 


 


 


อาจารย์ช่วยตอบด้วยนะค่ะ


ขอบพระคุณมากๆ ค่ะ


 



 

JY
09-08-2010 22:42:12
Addition of EtOH reduces polarity of the solution.  Also, EtOH, which can H-bond to water, then competes with DNA for water molecules.  This essentially strips off water from DNA, allowing for interaction with counter ions to neutralize the charges and reduction of DNA solubility.

Acetate is a weak acid with a pKa of around 4.75.  So, the solution containing sodium acetate is actually a buffer!
759
09-08-2010 22:59:08
Thank you. but I'm still confuse.
OK. If it is a buffer so the pH is not change much and what about reannealing of DNA?
tc
09-08-2010 23:47:50
ขอบพระคุณอาจารย์จิที่ช่วยกรุณาให้ข้อมูลครับ
กำลังเขียนตอบอยู่แต่เขาเรียก boarding แล้ว
เดี๋ยวพรุ่งนี้มาตอบเพิ่มให้นะครับ

ชอบคณะวิทย์ อย่าลืมมาเรียนเอกกับเรานะครับ (โฆษณาหน่อย)
tc
13-08-2010 19:30:02
ออภัยที่ให้คอยครับ  พึ่งจะมีเวลามา post คำตอบให้สมบูรณ์ครับ

Different kinds of salt (i.e. sodium acetate in this case) interact with the highly charged backbone of the DNA. It will, in effect, neutralize DNA and will make it ready for ethanol precipitation.

I would not say that adding salt causes DNA to re-anneal! I would say that it improves the stability of DNA double helix by reducing the repulsion between negatively charged backbones.

nchp
18-08-2010 10:51:24
 อ๋อ เหมือนกับที่ใส่ MgCl2 ใน PCR ซึ่งช่วยเป็น Cofactor ของ enzyme และก็ช่วย neutralize Pi backbone ใช่ไหมค่ะ   ขอบคุณนะค่ะอาจารย์ เข้าใจชัดทั้ง 2 ท่าน เข้าใจชัดขึ้นเยอะเลยค่ะ
น้องม.ห้า
18-08-2010 23:26:01
Mgไม่น่าเกี่ยวกับneutralize backboneนะ Mgมันน้อยด้วย
ในPCR basic bufferมีmonovalent cation อื่นอยู่แล้วลองดูจิ
tc
18-08-2010 23:57:10
น้องม.ห้า is correct!!!

เด็กม.ห้าสมัยนี้เก่งดี




ผ่านมา
23-08-2010 02:34:46
แล้วใส่ Mg ทำไมล่ะครับ
ผ่านไป
23-08-2010 02:46:46

ถ้าเป็นแค่ cofactor


แล้วบาง paper จะปรับ Mg2+ ทำไม


แล้วทำไมถ้าใช้ Mg2+ มากๆ แล้วจะได้ product เยอะแต่ไม่ specific ครับ



 

tc
23-08-2010 03:02:12
มันเป็นการเพิ่ม processivity ครับ  ที่กล่าวว่าเป็นแค่ cofactor นั้น เราคงบอกว่า "เป็นแค่" ไม่ได้ครับ เพราะ Mg มีบทบาทสำคัญมากในฐานะ cofactor ครับ

ถ้ามองกันในแง่ function แล้ว  Mg เป็นส่วนหนึ่งของ active site ครับ  มันเป็นโครงสร้างที่จำเป็นกับการ introduce nucleotide ตัวใหม่เข้าไปครับ  โดยโครงสร้างนี้มันจะ คล้าย ๆ กับ การที่เราเอานิ้วชี้กับนิ้วโป้งมาทำเป็นรูปตัว L ครับ  หรือก็คือ index-palm-thumb  โดย Mg อยู่เป็นแกน

ทีนี้เราก็ต้องมองว่าเอนไซม์พวกนี้ต้องท่องเที่ยวไปบนสาย DNA ครับ  ถ้ามันวิ่งดี (พูดง่ายๆ ก็คือมี processivity สูง) มันต้องมี abundant supply ของ nucleotide และ Mg ครับ  ทำให้เราต้อง vary Mg ให้เหมาะกับปฏิกริยาของเราครับ  ผมเชื่อว่าการที่เราใส่อะไรเข้าไปก็คงไปกระทบหลายปัจจัยครับ  ในแง่ของประสบการณส่วนตัวจากการทำวิจัย  ผมคิดว่าน่าจะมีผลต่อการท่องไปของ DNA polymerase ครับ เพราะ Mg มันช่วยมากใน template ที่ยาว (คนไหนช่างคิดหน่อย อาจจะมองกลับกันว่า เอนไซม์ไม่ได้เคลื่อน แต่ DNA เคลื่อนต่างหาก)


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