Week 6 - 2017

Analysis of recombinants via restriction mapping - Part 1

The final step when subcloning is to identify a clone containing the desired recombinant plasmid. In our case, we are also evaluating two different subcloning methods so we want to measure how frequently each method produced the desired recombinant.
There are several ways to evaluate the recombinant plasmids, we will use restriction mapping. Last week you decided how many colonies you wanted to screen for each method. This week we will select and grow the desired number of recombinants, isolate the plasmids and digest them with the appropriate restriction enzyme combinations. Next week we will complete the restriction enzyme mapping by running the digestion products on agarose gels.

Colonies need to be grown up in liquid culture in preparation for plasmid isolation. At least one person from each group needs to come in the day before your lab to inoculate cultures for this purpose.

Read/do before coming to the lab

  • The day before lab you must come in and inoculate your cultures. For the Monday lab, you will need to come in on Friday. Instructions for inoculating are below.
  • If you didn’t have time to plan the digests last week, do this before you come to lab. We need to check your plan before you start.
  • By this point, we expect that you can decide for yourself what you need to read before coming to the lab.

Protocols

  • Alkaline lysis & plasmid purification using EconoSpin columns.
  • HindIII and BamHI Fastdigest protocols from Thermoscientific. You looked up this information for Lab 1, please make sure you bring this information to today's lab. We are using the FastDigest enzymes.

To do the day before the lab:

Inoculate cultures for plasmid preps

  • count the colonies on each plate (note colour)
  • For each colony you want to screen, aliquot 5 ml of LB media (with 100 ug/ml ampicillin ) into a snap cap culture tube (use sterile techniques – if you do not know what this is, ask your instructor to show you).
    • The media has been prepared for you and should be on the bench.
    • Clearly label the tubes so you know which plate each colony comes from.
  • Circle the colonies you plan to use.
  • Touch each colony with the tip of a sterile toothpick and drop the toothpick into a culture tube.
  • Place the cap on the tube (to the first stop – the cap should be on but still loose – the bacteria need air to grow).
  • Place the inoculated tubes in a plastic 250 ml beaker and tape them in with masking tape (there should be a demo beaker sitting on the bench). Don’t forget to put your group number on the tape.
  • Your instructor will place the beakers in the 37 °C shaking incubator (~ 250 rpm) around 4 pm today. The beakers will be stored at 4 °C after 16 hr of incubation.

To do today:

  • Isolate plasmid DNA from 1.5 ml of each culture using the same protocol we used in week 1.
  • Set up restriction enzyme digests (you worked out what digests to do last week).

KEEP TRACK OF WHICH PLASMID GOES INTO WHICH DIGEST.

  • Incubated digests at 37 °C for at least one hour and then store at - 20 °C.
  • Count colonies on positive control plates and calculate transformation efficiency.
  • Make a table in your notebook indicating for each possible recombinant what sized fragments you should see after digestion. We will give you a template to follow in lab. Remember, you are doing two different digests so you need to make predictions for both. If you don't have time to finish this before the end of the lab, make sure your predictions are in your notebook when you hand it in.

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