Week 2 - 2015

Determine plasmid quality and concentration and do larger scale digests

Today we will be digesting pKC7 and pUC18 with the restriction enzymes BamHI and HindIII in preparation for recombination. 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 each plasmid, we need to determine the plasmid concentrations. We do this 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 can be determined by comparing the digested plasmid fragments to a DNA standard. The DNA standard consists of several DNA fragments of known size and amounts. DNA quality is determined by inspecting the resulting digest fragments to make sure we get the size(s) expected (what sizes do we expect for each plasmid?). Plasmid's are quantified by comparing the plasmid fragment's florescence to the florescence 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.

Before coming to lab this week you will need to

  • Complete all readings listed below.
  • Complete and submit assignment 2 via turn-it-in.com.
  • Print the product information sheets for FastDigestHindIII and FastDigestBamHI found at Thermoscientific (we will check that you have brought the correct protocol with you). Hint: look under resources once you get the page for the specific enzyme. Note that there are conventional versions of these enzymes, but we will be use the FastDigest versions.
  • Determine the expected DNA fragment sizes for the 2 digests we set up last week. Hint: reread the resource page About Plasmids and Vectors.
  • Read - working with DNA volumes and amounts - and try out the practice problems. We will be doing several calculations that involve working out what volume of a DNA solution will give a specific amount of DNA.

Read before coming to the lab

  • Project overview - Step III - Produce and prepare the DNA fragments for cloning.

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.

  • All protocols listed below.

Protocols

  • General guidelines for molecular biology labs
  • Protocol for Fast Digestion of Different DNA (this will be found in the product information sheets for FastDigestBamHI and FastDigestHindIII which you have already downloaded and printed.
  • Agarose gel electrophoresis of DNA
  • Tutorial - how to pour a gel
  • Estimating DNA fragment sizes on an agarose gel
  • Estimating DNA concentrations - agarose gel method
  • How much digested DNA do I need?
  • T4 DNA ligase; find this at Thermoscientific. We will not be setting up ligations this week, but we need this protocol to determine if our plasmid digests are concentrated enough.

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 ready for you when you come to lab.

Note: To calculate a precent-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)

  • For this first digest we will be loading the entire digest on an agarose gel. Work out how you will prepare samples for loading (we will check this before you start)
  • set up the gel running apparatus
  • load 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. The protocol How much digested DNA do I need? will help you work this out.

  • photograph gel
  • determine the digested plasmids’ quality (look for complete digestion and the correct sized fragments)
  • 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.

Set up a second set of digests

  • Follow the protocol "How much digested DNA do I need?" to determine the amount of pUC18 and pKC7 DNA you will need to digest (we will be doing this while the first gel is running).
  • Based on the required final concentrations needed for each plasmid, set up a second set of 20 µl digests with the appropriate amount of each plasmid.
  • Fill in the table below and have us check it before you start
  • Incubate digests as suggested by the enzyme manufacturer

NOTE: all volumes are in µl

component pKC7 digest pUC18 digest
DNA
HindIII
BamHI
10 X FastDigest Green buffer
H20
total vol 20 20

Run a second agarose gel.

  • Follow the same steps as you did for the first digest except this time only load 2 µl of each plasmid.

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.

Prepare for gel purification of Gene A: do you have enough digested pKC7 for gel purification?

Work out the total amount of Gene A you need to purify (see protocol book for how to do this).

  • Estimate the newly digested pKC7 concentrations
    • is there enough pKC7 to get the amount of the Gene A fragment needed for gel purification?

Prepare for the ligation reactions: is your vector (pUC18) concentrated enough?

We are using T4 ligase from Thermoscientific. We will be setting up 20 μl ligation reactions and will aim for the midpoint amount of vector (see the manufacturers product information to find the recommended amount of vector needed in each ligation reaction).

Recommended amount of vector needed in each ligation reaction =

You can only add 3 or 4 µl of digested pUC18 to the ligation – will your pUC18 concentration be high enough to get the desired amount of pUC18 in the ligation?

desired amount of pUC18 in each ligation reaction =

[digested pUC18] =

volume of digested pUC18 needed=

Preparation for shotgun ligation: what volume of digested pKC7 is needed?

For the shotgun ligation we will use 1:1 molar ratio of digested pKC7 to digested pUC18

desired amount of digested pKC7 ___ ng

[digested pKC7] =

volume of digested pKC7 needed to get the desired amount _ μl


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