Week 2 - 2017

Determine plasmid quality and concentration and double digest the plasmids

Today we will start preparing pKC7 and pUC18 for the ligation reactions by digesting them with the restriction enzymes BamHI and HindIII. For why we are using these enzymes see Project Overview Step III and take a look at these plasmid maps.

Before we can set up digests with the correct amounts of plasmids, we need to know the plasmids' concentrations. We determine these by digesting a small amount of the isolated plasmids with DNA restriction enzymes (digests were set up last week) and analyzing the digestion products using agarose gel electrophoresis. DNA quality and quantity is determined by comparing the digested plasmid fragments to a DNA standard (DNA ladder) that consists of several DNA fragments of known size and amounts. Plasmid quality is determined by making sure we get the expected DNA fragment sizes following digestion (what sizes do we expect for each plasmid?). Plasmids are quantified by comparing the plasmid fragments' fluorescence to the fluorescence of the DNA standard fragments. This process is described in detail in the protocols "Estimating DNA fragment sizes on an agarose gel" and "Estimating DNA concentrations - agarose gel method".

Using the determined concentrations we will then prepare digests with the appropriate amount of each plasmid for the next steps.

Read/Watch/Do before coming to the lab

  • Complete and submit assignment 2 (see lab schedule for submission details).
  • Complete the pre-lab quiz (due before your lab starts) - you will want to do all the indicated lab preparations before you attempt this quiz.
  • Print the product information sheets for FastDigestHindIII and FastDigestBamHI found at Thermoscientific (we will check that you have brought the correct protocol with you).
    HindIIIFastDigestManual
    Note, there are several Manuals and Protocols linked on the enzyme page. Please make sure you get the full product information for each enzyme (they are 4 pages long and are titled "Product Information").
  • Tutorial - how to pour a gel
  • Read

It is a good idea to review ALL project steps each week before coming to lab. This will help you get a better handle on the whole project as we move along.

  • Try out the practice problems for DNA volumes and amounts. We will be doing several calculations that involve working out what volume of a DNA solution will give a desired amount of DNA.
  • Determine the expected DNA fragment sizes for the 2 digests we set up last week. Hint: reread the resource page About Plasmids and Vectors.
  • Prepare to use the protocols listed below.

Protocols

  • General guidelines for molecular biology labs
  • Protocol for Fast Digestion of Different DNA (this is found in the product information sheets for FastDigestBamHI and FastDigestHindIII that you have already downloaded).
  • Agarose gel electrophoresis of DNA - note there is a lot of information in this protocol - you need to read all the information - but pay particular attention to the "Quick Guide"
  • Estimating DNA fragment sizes on an agarose gel
  • Estimating DNA concentrations - agarose gel method
  • T4 DNA ligase; find this at Thermoscientific. We will not be setting up ligations this week, but we need this protocol to determine how much plasmid to double digest.

To do today:

Run agarose gel

  • A prepared 25 ml, 0.9% agarose gel (1X TAE) with 0.1 µg/ml ethidium bromide, will be shared between 2 groups.

Note: To calculate a percent-weight-per-volume (agarose is a solid and is weighed but TAE is a liquid), calculate the % of the volume in ml and convert to a gram weight (e.g. to make 1% gel in 100 ml you would use 1 g of agarose)

  • Prepare your digested DNA for loading. For this first gel, we will be loading the entire digests from last week - you worked out how to prepare for loading last week so that we can load these gels without delay today!
  • Set up the gel running apparatus.
  • Load the gel with 5 µl of prepared 1KB DNA ladder (50 ng/µl) and each groups’ prepared digest samples (place the ladder between the 2 groups samples)
  • Run gel for 30 – 50 min at 70 volts.

While the gel is running, prepare a second agarose gel and work out the amounts of pKC7 and pUC18 that you need to digest in order to have suitable concentrations for the gel purification and ligation steps. See below.

  • photograph gel
  • Determine the plasmids’ quality (look for complete digestion and the correct sized fragments - you determined the expected sizes before you came to lab)
  • Determine the digested plasmids fragment sizes and the concentrations of each digested plasmid. Remember to record this in your notebook, and explain how you arrived at each estimated value.

Determine the amounts of DNA needed for each recombination

Figuring out how much of DNA we need to digest requires working backward from the amount needed in each ligation reaction.

  • Consult the manufacturer's ligation protocol to find out the reaction volume and how much DNA is needed (you brought this protocol with you before lab). Generally, they will suggest a range of vector and insert amounts; depending on your particular application, you may need to do some empirical testing to get a good result.
  • For both the targeted and shotgun strategies, we are going to aim to use 50 ng of digested vector (pUC18).
  • For the targeted ligation, we want to use a 3 molar ratio of insert to vector. Use the following to calculate how many ng of the GeneA insert will give us 3 times as many insert fragments as pUC18 fragments.
calculate-insert-amt.png
  • For the shotgun ligation, we are using a 1:1 molar ratio of vector plasmid to inserts. Calculate the amount of digested pKC7 needed in the shotgun ligation by inputting the entire size of the pKC7 as the insert.

How much plasmid to digest?

The above calculations tell us the amount of DNA needed in the ligations, but since the digests and the ligations are done in solution, what we really need to know is the volume of DNA needed. For example, we need 50 ng of digested pUC18 for the ligation step, but if we are doing a 10 µl ligation reaction, how many µl of pUC18 do we want to add and will this volume "fit" in our reaction? We need to calculate the minimum DNA concentrations needed in order to "fit" all the components in the reaction.

  • From the manufacturer's instructions, what is the ligation reaction volume and how much "room" is left after you add the required components (e.g. the ligase)?
  • pUC18 digest: we will use the same digested pUC18 for both ligations and we would like to add only 1 - 2 µl of digested pUC18 to the ligation. How many ng of pUC18 will we need to add to a 20 µl digest so that the pUC18 concentration is sufficient for our needs?
  • pKC7 digest - targeted ligation: this is a more complex calculation because we need to first gel purify the GeneA containing fragment. Yields from gel purification are only 50 - 80% (aka if we put 100 ng of DNA on the gel, we will only get 50 - 80 ng back). Additionally, we will be eluting the purified fragment in 20 µl, so if we put 100 ng on the gel and we got an 80% recovery, the concentration of the purified fragment would be 80 ng /20 µl = 4 ng/µl. To get the 100 ng into the ligation reaction, we would need to add 25 µl of the purified fragment; we would not have enough purified fragment and it would be too dilute to "fit" into the reaction.
    • A better way to do this is to determine the minimum concentration needed. Assuming we do not add more than 2 µl of digested pUC18 to the ligation reaction, what is the maximum GeneA volume we can add?
    • Based on the maximum volume we can use, what is the minimum GeneA concentration needed?
    • Knowing that we are eluting in a 20 µl volume and accounting for a 50% loss during the gel purification process, how much GeneA needs to be loaded onto the gel for gel purification?
    • Finally, how much pKC7 needs to be digested to get the desired amount of GeneA?

Double digest pKC7 & pUC18

  • After determining the plasmid concentrations you isolated last week, work out the amounts of each component needed in the double digests and fill in the table below. We will check it before you start.
  • Incubate digests as suggested by the enzyme manufacturer.
component pKC7 digest (µl) pUC18 digest (µl)
DNA
HindIII
BamHI
10 X FastDigest buffer
H20
total vol 20 20

Run a second agarose gel.

  • After the digest is complete, follow the same steps as you did for the first digest but this time only load 2 µl of each plasmid.
  • While the gel is running
    • work out what sizes you expect to see on the gel
    • complete your notebook. Make sure you fully explain the rationale behind the amounts of plasmid you are adding to each digest. In your second digest table, make sure you indicate both the volumes and the amounts of plasmid you added to each digest (this will save you time later in the semester).

Your notebook must contain both gel photos obtained today and a full analysis of both gels (aka - did you obtain the expected fragment sizes and what are the DNA concentrations of the plasmids you isolated last week, and what are the concentrations of the double digested plasmids).

The plasmids must be completely digested and sufficiently concentrated for the steps we will carry out in the next few weeks; therefore, if you have any incomplete digests this week (or any other problems with your digested plasmid DNA) we will need to fix the problem before next week. The lab instructor will go over your results with you today and give you direction on what to do if there are any problems. Fixing the problem may involve coming back outside your scheduled lab time.


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