Services | Minipreps (+DNA Sequencing)

What customers can provide:

• Plasmid DNA - Please review our Complete Mini-Prep Service below
• Bacterial colonies on a plate - Please review the information provided below
• Inoculated cultures to be grown - Please review the information provided below
• Spun-down pellets - Please review the information provided below

Labelling Samples

-- Please provide your samples in tubes labeled sequentially from "1" onwards

-- As customers’ samples will be provided back in much smaller tubes than the culture tubes, excess text will hamper accurate transfer of sample name from one tube to another

E. coli Strains

• We have found that DNA isolated from those E. coli strains in which the Endonuclease A has been mutated, results in purification of DNA that is more fully intact (non-degraded)
  • Please consider this should you choose to send us starter cultures/spun down bacterial pellets
• Please note we only use the DH5alpha E. coli strain in-house

Media & Growth Considerations

• Media Consideration: When growing your cultures, please use only LB broth and a volume of no more than 5ml
• Time Considerations: We have found that conditions that favour plasmid isolation include those where growth of the culture does not exceed 16 hours
• Using too large a volume, an inappropriate growth media (ie. TB broth) or growing for too long results in a lot of bacteria but not much plasmid
  
  • Ultimately, excess bacterial cell material can clog up the clearing filter and leads to loss of your plasmid material

Bacterial Colonies on a Plate

• After you have transformed your plasmid(s) of interest, you can send us your plate(s) and our facility will:
  1. Select 1 colony per plasmid
  2. Grow mini-prep cultures
     • Please inform staff about antibiotic resistance for each plasmid
  3. Isolate DNA

Inoculated Culture To Be Grown

• Starter Culture: Please send your starter culture (5ml) in which you have placed a single bacterial colony, together with details of the volume of culture and antibiotic to use
• Antibiotic: As standard we have -
  • LB + Ampicillin
  • LB + Kanamycin
  • If you require a different antibiotic, we ask you to please grow your own culture and provide these to the DNA Sequencing and Services facility
• Our facility can grow your miniprep and isolate DNA directly from each culture  

Spun-Down Pellets (Centrifuged Samples)

• When supplying us with your pelleted bacteria, please ensure that they are in Falcon 2059 tubes.
  
  • We are unable to accept samples in other types of tubes, since these do not fit on our robots.
• To get the best results, ensure that you drain off all the culture medium, since not doing so reduces the quality of the plasmid DNA produced.

Complete Mini-Prep Service

Should you wish for DNA Sequencing and services to take care of your miniprep from start to finish, we can easily do so. Please provide samples of DNA and we can take it from there! We don't stipulate an exact amount of DNA, but if you send us 20ul of your plasmid at 10ng/ul, that will be enough. We will transform your DNA, pick two independent colonies to grow cultures, and isolate the DNA for you.

As standard, we have LB medium plus ampicillin and LB plus kanamycin. If you require a different antibiotic, you will have to grow your own culture.

Having Your Miniprep Sequenced

It is often important to confirm the sequence of one's insert before embarking upon additional planned research endeavors in the lab. This can easily be accomplished by sequencing the DNA template which you have just had prepared by us. Simply click "Miniprep + DNA Sequencing" above.

During the submission process, we will request additional information that will allow our facility to carry out this sequencing service for you including:

• Information regarding your primer
  • We have a number of Standard Primers available
  • Or you can provide your own at the time that you submit your miniprep sample
• Whether your template is known to be GC-Rich

Please contact us if anything is unclear!

Safety

• As with all our services where we are accepting live bacterial material, please note that we can only accept samples that have been classified as biohazard containment level 1 according to the UK Genetically Modified Organisms (Contained Use) Regulations and ammendments.
• For more information on these regulations, please see the HSE website.
• We may require proof of containment level to be supplied.

DNA Isolation

• MRC PPU DNA Sequencing and Services utilises Promega Wizard SV96 Plasmid Purification System™ to prepare DNA that is ready for your downstream applications.  
• DNA is supplied in distilled water direct from the robot with no additional characterisation.

Quiagen Robot

• This efficiency, and the associated manner in which the robot functions, does mean that there is the small possibility of carry-over of plasmid from one sample to another. This is inherent to the robot but can be minimized.  
• We would like to ensure that customers have read the disclaimer below regarding this, which includes steps that can be taken by customers to minimize downstream errors. 
• Please let us know if you have any questions.

Potential Yield of Plasmid DNA

• Please note that the quantity of plasmid recovered from a mini-prep is dependent on many factors and not just the size of the pellet or how cloudy the culture looks
  • MRC PPU DNA Sequencing and Services cannot currently quantitate the resultant DNA from each customer's prep 
• Plasmid Copy Number Considerations 
  
  • High Copy Plasmids
    • Given the nature of the replication process of high copy plasmids, these will generally yield more copies and thus more DNA from mini-preps than low copy plasmids
  • Low Copy Plasmids
    • When using low copy plasmids, please ensure that you have identified the optimal strain to use for plasmid growth prior to submitting your cultures

Disclaimer Regarding Sample Carry-Over

The Qiagen Bio-Robots that we use to perform bacterial DNA mini-preps are designed for rapid isolation of the DNA and the usage of a minimum amount of consumables. For this reason, they are equipped with stainless steel probes that are used for reagent dispensing AND sample transfer. Although there are multiple wash steps using both water and chemical washes, some very minor carry-over of material from one prep to another WILL occur. This has been checked and we have taken every step to minimise this carry-over. However, we cannot eliminate it.

This carry-over will not affect any standard diagnostic or analysis step performed on the mini-prep DNA (e.g. restriction digests, sequencing, etc). However, if you subsequently re-transform the mini-prep DNA and pick a colony, there is the small chance that the colony chosen might represent mini-prep DNA that was carried-over from a previous sample. As with any good laboratory practice, we also encourage customers to check their mini-prep plasmid prior to generating a larger preparation of DNA -- even if they are not using a mini-prep protocol that introduces some carry-over.

As the carry-over discussed above is inherent to the robots used in our service, MRC PPU DNA Sequencing and Services is not responsible if customers neglect to check and confirm the identity of plasmids used for follow-on studies.

Below we have provided additional information regarding ways to overcome carry-over issues:

Replica-plate before mini-prepping

When picking a colony from a transformation, streak it onto a new agar plate using a reference grid and then use the same colony picker to start the mini-prep. In this way, you can go back to the replica streak once you know that the mini-prep is what you want.

Replica-plate after re-transformation

If you decide not to replica-plate at the start, you can do this when you re-transform the mini-prep DNA for a larger prep (e.g. maxiprep). In this case, simply transform bacteria, plate these out and pick a number of colonies (e.g. six). Streak these onto a new agar plate and use the same colony picker to start a mini-prep. Once you have performed the mini-prep and know which colony is right, you can go back to the replica-plate for setting up the maxi-prep.

Which Method Is Best?

Of the methods outlined above, there is no 'best' one. Rather, we encourage customers to consider their cloning strategy, number of colonies needed for initial screening

• *Quant-It: We are happy to quantitate the concentration of your template for only £1
• ^Augmented Protocol: If your template has known secondary structure or a high GC content, you can select our augmented protocol - £0.50 will be applied per reaction to facilitate this.

Do you routinely need a large number of mini-preps prepared and sequenced?

Why not contact our staff to learn about savings options?

Our pre-payment option will help you save more per reaction!

Primer

• Standard primers offered by MRC PPU DNA Sequencing & Services are free!
• Custom primer synthesis will reflect the size of the primer – the cost to the customer is £0.50 per base

Learn more about all our pricing and turnaround.

Please note that prices are exclusive of VAT, which customers outside The University of Dundee will be charged at the appropriate rate (currently 20%) on all products and that all sales are subject to MRC PPU DNA Sequencing and Services' Terms and Conditions.

Many of our customers prefer to take care of the steps involving choice of bacterial colony and growth of culture. Thus from the point of a centrifuged pellet, our robots routinely purify 96 bacterial plasmid DNAs in two hours with minimal intervention from staff.

We are happy to assist with the additional steps outlined above – from initial transformation of plasmids, all the way through to DNA Sequencing. The time involved for all these steps will increase accordingly but we are happy to communicate with customers so that they are apprised of timelines throughout this process.

Shipping Details

• Labels
  • Please ensure that samples are clearly labelled so that we can identify them.
• Packing Materials
  • Eppendorf Tubes (if sending plasmid DNA to be transformed)
    • Please also ensure that your samples are well protected and packaged to meet Post-Office or courier regulations
      • A "Jiffy" style padded envelope for DNA samples in plastic tubes works well
      • We discourage use of regular envelopes as in the past, we have received envelopes with tears and/or crushed tubes in them
  • E. coli Pellets
    • None needed if hand delivering (internal customers)
    • Packing in dry ice within a polystyrene insulated container is the only packaging method we can accept for items sent by courier

Postage Costs

• Customers are responsible for paying all postage / carriage charges
  • For DNA samples or agar plates, normal first class post should be sufficient
    • As this does not always arrive as "next day post", we recommend that customers who have deadlines to meet send their samples by a guaranteed next day delivery service
• Insufficient postage provided on a package will cause a delay in use receiving the package
  • If MRC PPU DNA Sequencing and Services is then charged upon receipt, please note that we will need to pass this cost onto the customer