Week 7 - 2016

Analysis of recombinants via restriction mapping - Part 2

Previously we used the restriction maps for the vector and insert DNA to determine the fragments we should see when we digest the new recombinants with BamH I and Hind III.
Last week you digested several recombinant plasmids with these enzymes.
This week you will identify the recombinants you created by separating the digested recombinant plasmid fragments on agarose gels.

To do before the lab

  • determine the expected banding pattern each possible recombinant will give in each digest condition
  • work out how you will prepare your samples for loading - the quicker the gels get loaded the sooner they will be running and the sooner we will be done!
  • the day of lab each group needs to come in a prepare a 30 mL agarose gel
    • use 15 well combs instead of the 8 well combs
    • 0.9% agarose in 30 ml of 1X TAE - add 5 uL of ethidium bromide
    • gels must be poured the day of your lab between 8:30 am and 2:00 pm - please sign up for a time slot on UR Courses.

In lab

  • separate the digested recombinants on agarose gels
    • everyone will use the same loading order on their gels - this will be given to you in lab
    • for each sample load the entire digest on the gel – see Quick guide to preparing and loading samples
    • also load 5 µl of DNA ladder (use the same ladder prep as used in previous weeks)
    • run the gels at 80 volts for at least 60 min (we need excellent separation to get good results)
  • while the gels are running, we will talk about the lab report
  • photograph and analyze the results

Remember when doing the analysis you already know what size fragments you expect to see – use this information to clearly identify each band’s size and to identify the recombinant.

If there are any problems with your results (incomplete digests, mixed up samples etc.) you will need to come in and repeat the plasmid preps &/or the digests on your own time – before the end of this week. Discuss any problems with Lauri ASAP.

  • before leaving the lab today, your group must hand in a full analysis of all the recombinants – there will be a spreadsheet to fill in on UR courses
  • all results (plate counts, gel photos and recombinant identities) will be posted

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