MiniPrep problems

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Pinal Pandya
Pinal Pandya's picture
MiniPrep problems

Hi all,
I have been trying to isolate plasmid from BL21. I also used to isolate the plasmid from DH5alpha. But both times i just dont get either the right bands or any plasmid at all. I have used the qiagen kit. Also i am trying to use home made buffers from Molecular cloning book. I follow the protocol to the tee. so i dont understand why i am facing so many problems. please help me!!!!

thanks
Pinal

Shubhangi
Shubhangi's picture
Its unlikely not to get a

Its unlikely not to get a proper result even after using a kit.
Check below mentioned things

  1. Expiry date of the kit you are using
  2. Number of cells you have been using.
  3. Confirm proper cell lysis- viscous sticky suspension  should be observed after completion of lysis
  4. Column filters are intact i.e. no cut or damage to the column.
Arvind Singh Pundir
Arvind Singh Pundir's picture
Pinal Pandya wrote:Hi all, I

Pinal Pandya wrote:

Hi all, I have been trying to isolate plasmid from BL21. I also used to isolate the plasmid from DH5alpha. But both times i just dont get either the right bands or any plasmid at all. I have used the qiagen kit. Also i am trying to use home made buffers from Molecular cloning book. I follow the protocol to the tee. so i dont understand why i am facing so many problems. please help me!!!! thanks Pinal

 
try again with the comments of subhangi with QIAGEN KIT
also go to the following link hope you will find your querry in any of the suggestions
http://www.protocol-online.org/biology-forums/posts/12277.html
 
 
good luck

Kvachhani
Kvachhani's picture
Try it with "Bangalore genei"

Try it with "Bangalore genei"  or sigma kits.

Jason King
Jason King's picture
If your bacteria are growing

If your bacteria are growing up well under antibiotic selection then you should have plasmid in the cells. Poor results in Qiagen preps (or any other brand kits) are usually due to overloading of the columns - especially when there is a low copy number (large amount of biomass).
My advice would be to use the Qiagen solutions P1, P2 and P3 (or if you make them up yourself, check that you have done so correctly by checking the recipe which is in the Qiagen handbook), but not the columns.
 
ie. Do P1-3 then centrifuge fast.
Transfer the clear supernatant to a fresh tube
Add 0.7 volumes of isopropanol, mix well
Centrifuge fast for 30 mins
Remove and discard supernatant
Wash pellet with 300 microlitres of 70% ethanol
spin for 10 mins fast
remove supernatent, dry pellet
resuspend in 50 microlitre of Tris or water (as you like)
These samples should contain plasmid. If after RE digests they look strange then it could be that there is something being expressed in the coli that is toxic to them, leading them to select for wierd mutants (i have some experience in wierd mutants!)

RLS
RLS's picture
 What vector are you trying

 What vector are you trying to isolate (what is the backbone, ie, puc18, pET15b, etc.) ? Is it a low copy number plasmid?

Rajeshwari patel
Rajeshwari patel's picture
Hi, Pinal.

Hi, Pinal.
First you check Your antibiotic pressure( means antibiotic condition )which you are using to grow the E Coli, If it is correct than kit and than you check water or buffer whhich you are using for dissolving the DNA
 
I think You will get the  results soon.

RLS
RLS's picture
The antibiotic issue is a

The antibiotic issue is a very good point. You do want to make sure your antibiotic is fresh and functional. Have you had any luck getting this worked out?