Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > DNA Techniques > DNA Extraction Forum
Register Search Today's Posts Mark Forums Read

DNA Extraction Forum DNA Isolation Forum. A forum specifically about questions on DNA extraction and purification.


Plasmid DNA extraction help

Plasmid DNA extraction help - DNA Extraction Forum

Plasmid DNA extraction help - DNA Isolation Forum. A forum specifically about questions on DNA extraction and purification.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 11-21-2008, 02:23 PM
Pipette Filler
Points: 222, Level: 4 Points: 222, Level: 4 Points: 222, Level: 4
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Nov 2008
Location: Madeira Island
Posts: 10
Thanks: 0
Thanked 0 Times in 0 Posts
Smile Plasmid DNA extraction help



Hello!

I am new here! My name is Michael, i have 22 years old and i'm on the last year of my biology course.

I found this forum while i was searching for some answers regarding Plasmid DNA extraction, and i though you could give me a little help interpreting the results, as this is my first time with DNA extraction.

The pictures i show bellow are all from the entire class, but what is most interesting, is that although all of us followed the exact protocol, the results were totally different!

The bacteria we used to extract the plasmid DNA were previously grown in LB medium with 5L ampicilin.

The three buffers we used during the extraction were:

Buffer 1-
50 mM Tris HCl
10mM EDTA
100 g/ml RNase

Buffer 2-
200M NaOH
1% SDS

Buffer 3-
3M potassium acetate (pH 5,5)

The samples of each class group were run in 1% agarose gel (TAE 1X and 5L Ethidium bromide).

The sample buffer was composed by
0,2% bromophenol blue
0,2% xylene cianol
60 mM EDTA
60% Glicerol

Well, and now the worst part, the results (all of the same gel taken on 2 different stages, 2 with white background and 2 in black)









The sample in well number one is the DNA marker. The rest are samples from each student (mine is sample number 3)

So based upon this, i would like to hear your comments.
I am wondering why, in sample 3, i got some bands that are "above" the first band of the marker. IS this normal?
And regarding the bottom huge roudish bands that most people got?
In your opinion, wich sample has more DNA (if any is present)?
And finally, about well number 7, why are the bands so weak?


After this, we also did DNA restriction with 3 enzymes ( BamHI, SacI and EcoRI. ) and used 10X FastDigest buffer. We used 1 enzime unit, during 30 minutes, at 37C (agarose gel at 0,6%)

These are the results we obtained yesterday:




M - Marker (i will post latter wich marker we used, as i do not remember right now)

E- Eco
B- Bam
S- Sac

In the end i will need to build the restriction map to my report, but at this point i am lost. I apologise if any of my questions might sound stupid or inapropriate or if the answer is pretty obvious, but i still need to get used to this and also want to be 100% sure of my conclusions.

Thanks for any help!

Regards
Michael
Reply With Quote
  #2  
Old 11-21-2008, 03:06 PM
admin's Avatar
Administrator
 
Join Date: Nov 2005
Posts: 1,418
Thanks: 883
Thanked 68 Times in 58 Posts
Default Re: Plasmid DNA extraction help

Hello Michael,
welcome to the site and forums.

EDIT: actually before I analyze this more, can you post the DNA Marker sizes for me please? thanks

I enjoyed my molecular biology classes they were a puzzle... and I see you enjoy yours

I will try to help. Your band in the 3rd lane I assume is plasmid not genomic DNA which good, although less yield than some of the other lanes. Make sure you compare your band with the ladder sizes to see if its in the right size. Also as you mention there are higher bands, I will get to both points in a second.

Good isolation of DNA usually yields higher bands equivalent about to the size of the plasmid not a lot lower (genomic or chromosomal DNA if you are doing a plasmid extraction, degradation products of the genomic DNA, fragments of genomic DNA) bands.

However also, as you may know there is supercoiled, nicked and "normal" plasmid DNA which may be seen in your gel which will make it tough to estimate this, and thats why they make you cut your DNA (which removes the coils and nicks) and the size of this cut DNA should be almost exactly the size of the plasmid from the bacteria you grew.

Furthermore, depending on the isolation/purification and the user, there may be DNA binding proteins (not that good) in your sample that keep your DNA up high, or the DNA may be aggregated with other nasties (membrane, viscosity or other factors keeping it in the well and preventing it from running into the gel) thus keeping it in the loading well. You may also have contaminating genomic DNA or even RNA (you would see this much lower in the gel as a streak or 2 bands corresponding to the ribosomal RNAs (16s? and 23s?? i may be wrong in the numbers its been a while).

Did you use RNAses to remove the RNA or a method to separate out RNA from DNA?

Also how did you separate out the plasmid DNA from the genomic DNA of the bacteria? Usually the genomic DNA of bacteria is connected to the membrane, whereas plasmid DNA is in the cytoplasm so if you are careful in lysis they won't get mixed

Let me know if you have any more questions, I enjoyed the pictures.

cheers

Last edited by admin; 11-21-2008 at 03:16 PM.
Reply With Quote
  #3  
Old 11-21-2008, 06:18 PM
Pipette Filler
Points: 222, Level: 4 Points: 222, Level: 4 Points: 222, Level: 4
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Nov 2008
Location: Madeira Island
Posts: 10
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: Plasmid DNA extraction help

Hello again, and many thanks for the reply!

Yes, i really enjoy these classes! It's so exciting to work with DNA, such a fragile and at the same time, complex chain!

Our objective was the extraction of the plasmidic DNA, so those are great news indeed! We used a plasmid called pAc-PAL (1,015kb + 398pb).

I will quickly try to resume what we did
To separate the RNA from the DNA, we used an RNase on buffer number one. After applying this buffer we left the solution 5 minutes at environment temperature to digest the RNA, then we added buffer number two (wich has NaOH who will break the cell walls and membranes and release the cell contents ) Quickly after we add the buffer number two, we left the bacteria at -20C during exactly 4 minutes (probably to make the lysis of the bacteria more efficient??), then we add buffer number three wich contains potassium acetate. I made a search regarding this compost and it seems to neutralize the alcaline lysis produced by the 2nd buffer. Besides that it also makes the proteins to precipitate, and helps only the plasmid DNA to renaturate again. The genomic DNA cannot renaturate, because it hasnt the ability to do it (that's what the book said about it). So after this we had centrifugated for about 20 minutes at -20C and then only collected the watery phase (we discarted the bottom sediment). This is how we separated the plasmid DNA from the genomic one. I hope you understand my english

Now regarding the supercoiled, nicked and "normal" plasmid DNA you mentioned, i also think you are totally right, I was expecting to get only one band, but probably the lighter bands we can see in some samples were vestigial remains of supercoiled and nicked DNA that didnt got cut off...


About the DNA marker sizes you asked me, i only know the sizes of the second gel (the restriction with enzymes). Regarding the first gel, my teacher only asked us to comment the results based only upon the bands and their relative position on the gel.

But regarding the restriction, these are the values of the marker:

21226
5148
4973
4268
3530
2027
1904
1584
1375
945
831
564

Now here i get another problem. We only have 12 values and we obtained around 17 bands (these are the bands i considered from top to bottom: 1 very large + 3 very weak + 2 large + 3 very weak + 2 bands side by side + 2 more bands + 3 lighter bands at the end.

Now i am trying to correspond each value to one band, and trying to discart 4 of the weaker ones, and above all, trying to get the distances proportional to the value of the marker... Can you give me some hints? Wich bands would you discart?

Thanks again
Michael
Reply With Quote
Reply

Tags
dna , extraction , plasmid


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
Problem in plasmid extraction Alston DNA Extraction Forum 4 07-17-2012 07:19 PM
[Yeast] 2 micron plasmid extraction Ellie Harrison Yeast Forum 3 06-18-2012 06:45 AM
Plasmid DNA Extraction charumathi DNA Techniques 3 02-17-2011 10:32 AM
dcm-,dam- plasmid extraction ese DNA Extraction Forum 0 06-29-2009 08:52 AM
Plasmid Extraction from Bacteria admin DNA Extraction Forum 2 06-29-2009 01:45 AM


All times are GMT. The time now is 07:25 AM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.14650 seconds with 16 queries