Bio 6B Lab Guide, Winter 2020

This lab guide is for the Winter 2020 course, which was taught on campus. For Spring 2020, Bio 6B (like all De Anza courses) will be taught online only. There will be no physical labs, so this lab guide does not apply. I'm leaving this page online for reference only.

This page is intended to help you prepare for each day’s lab. Many lab days will require you to read more than one page on the 6B lab site, so use this lab guide to tell you what you should read and to help you plan your lab work for each day.

This page will be updated as we progress through the quarter, so check it before each day's lab.

For an overview of the whole quarter’s activities in lecture and lab, see the 6B Calendar page.

Some gel photos will be posted on the Bio 6B flickr site.

Tuesday, Jan 7

On the first day of lab, you'll start to grow some bacterial cultures that you will use for the first six weeks of the quarter as you do the Conjugation and pGLO lab experiments. The procedures for the first day are simple, but it's important to start building your understanding of the concepts behind the experiments.

Reading on the 6B site:

Bacterial Strains & Plasmids

Starting Bacterial Cultures

Thursday, Jan 8

Use your bacterial cultures from the previous lab to begin two related multi-part lab experiments involving plasmids: Conjugation and pGLO. Since these two experiments overlap so much, I'm going to call them the plasmid labs.

Reading on the 6B site:

Conjugation.

pGLO Transformation.

Tuesday, Jan 14

You have several things to do for the plasmid labs. Make sure you do them all.

Look at your conjugation plate results

Conjugation. Did conjugation occur? What possible results could tell you that conjugation did not occur? If it didn't occur, how can your plate results tell you what went wrong? Remember the possible problem of satellite colonies. How can you be sure you're not looking at satellite colonies? Which colonies on your plates contain the plasmid?

Look at your pGLO transformation results

 pGLO: Plate results. Your plates should answer several questions:

  • Did the cells get transformed with pGLO?
  • Did arabinose induce the expression of GFP in transformed cells?
  • Did glucose inhibit GFP expression?

 Think carefully about how you know the answers to each of these. For each question, there is an experimental plate and a control.

Total nucleic acid extraction

This is a way of checking whether connjugation worked by examining the DNA. You should be able to find chromosomal DNA in all your cultures, but not all of them contain plasmid DNA. You'll extract plasmid and chromosomal DNA together (total nucleic acid) and run the DNA on an electrophoresis gel to find out which cultures from the conjugation lab (S17/pARO180, HB101, and conjugated HB101/pARO180) contain the plasmid.

Total Nucleic Acid protocol. You'll do the extraction today and the gel next time.

Thursday, Jan 16

DNA electrophoresis

See the electrophoresis page for background information.

See the DNA gel method page for step-by-step instructions.

Run the gel for your Total Nucleic Acid. Start the gel first, because it will take some time.

Start liquid cultures

You'll need liquid cultures of  both pGLO and pARO180 for Plasmid Purification. It's important to make sure you're actually culturing the cells that contain the plasmid. How will you know?

You restreaked some colonies from your original plasmid plates onto new plates. The purpose of that was to make sure that you have plenty of fresh colonies to work with later. Now use your restreaked plates to make two liquid cultures in LB/amp:

  • pGLO: use  a colony from the restreaked amp plate (or any other pGLO plate with ampicillin).
  • Conjugated HB101/pARO180: use a colony from the restreaked amp+strep plate.

When you leave lab today, you should have two liquid cultures in the shaker incubator: one with pGLO and one with pARO180. Both  cultures should contain ampicillin to ensure that only cells containing the plasmid will grow.

Some gel results from the total nucleic acid procedure:

IMG 0010a

 

IMG 4639a

Overall, everyone succeeded in extracting nucleic acids from their cultures and visualizing the DNA and RNA on the gel. However, the plasmid DNA turned out to be elusive; it was only visible in a few of the lanes where it was expected. The fact that we couldn't see it isn't evidence that it wasn't there; we need to move on to plasmid purification, which will give us a better way to see the plasmid DNA.

Tuesday, Jan 21

pGLO and Conjugation: Plasmid Purification. Use your liquid cultures to prepare samples of purified plasmids (pGLO and pARO180).

Thursday, Jan 23

This lab is about investigating your purified plasmid DNA (both pGLO and pARO180) by cutting the DNA with restriction enzymes and analyzing the cut fragments on a gel. There are two parts to this: a virtual digest and a real restriction digest.

Virtual digests

Virtual digest: you use a computer to analyze a plasmid DNA sequence to predict how it will be cut with restriction enzymes. This is an online assignment that you'll complete in lab, working together with your lab group. It will be much easier if you have a laptop (or at least if someone in your group does). You'll need to do some cutting and pasting and typing, which will make this assignment harder to complete on a phone or tablet without a keyboard.

Real digests

You'll do the real restriction digests next time, but you have some preparation to do today. Perform DNA assays on your plasmid DNA samples, then use your measured DNA concentrations and do the calculations for setting up the digests. If you get that done today, you'll be able to quickly set up your digests next time.

Tuesday, Jan 28

Restriction digests.

Thursday, Jan 30

DNA gel to analyze your plasmid restriction digest results. Prepare your gel with 1% agarose. Are you able to tell which plasmid is which from the gel results, even if the lanes aren't labeled?

Start liquid cultures for HIC and the protein gel, according to this table:

  Plasmid Arabinose?  Ampicillin?
 1  pGLO Green  +  +
 2  pGLO Green  –  +
 3  pGLO Blue  +  +
 4  pGLO Non-F  +  +

Tuesday, Feb 4

pGLO: Perform HIC. You should have four liquid cultures from last time, as shown in the table. Today you should make lysates from all four samples, following the protocol on the HIC page. You'll use two of these lysates today for HIC, and then use all four of the lysates later for protein electrophoresis.

  Plasmid Arabinose?  Ampicillin? Make lysate Perform HIC
 1  pGLO Green  +  + yes yes
 2  pGLO Green  –  + yes no
 3  pGLO Blue  +  + yes yes
 4  pGLO Non-F  +  + yes no

 Save all your lysates and HIC samples in the freezer at the end of lab today.

On the HIC page, I described the procedure as if you're going to do HIC with your +arabinose and -arabinose samples; in fact, you should do HIC with your tubes 1 and 3: green fluorescent and blue fluorescent.

Thursday, Feb 6

Protein assay

Prepare to analyze your protein samples from HIC using electrophoresis. Today you should assay your protein samples and calculate how  to load your gel. You'll run the gel next time.

Problem set

In-class group problem set: Open Reading Frames in pGLO.

Tuesday, Feb 11

You'll run a protein gel. Before you can load the gel, you'll need to figure out how much of each protein sample you're going to load. You should get this done before you come to lab; otherwise, you might have a hard time completing the experiment.

This gel has one main purpose: to find GFP. You should be able to find the GFP band using two criteria:

  • GFP should be present in the +arabinose lanes but not the —arabinose lanes.
  • You know the molecular mass of GFP, since you calculated it in a previous lab. Since GFP is relatively small, don't run your gel too far, or it could run out the bottom of the gel. Stop your gel when the lower blue line is still 5 mm or so above the opening in the gel plate.

In addition, the gel should reveal the other proteins produced by  your cells, and you may see some other differences among the different samples.

Plan the lab work

We have two different kinds of protein gels to use. I will give you one, and you should team up with another group to run your samples and their samples on your gel. Figure out which gel you're using and make sure you know which buffer and sample preparation protocol to use, and follow the instructions on the SDS-PAGE Method page.

Gel loading

There are 10 lanes on the gel. You should fill them all, because leaving empty lanes on a thin protein gel causes the bands to spread unevenly (this doesn't happen on the thicker agarose DNA gels).

For each sample (not including the marker lanes) you should load the maximum amount that will fit in the well. For the SDS-PAGE denaturing gel, this is 25 μl. For the non-denaturing PAGE gels, it's 40 μl.
 
Load your gel like this:
 
  1. Mark 12 Protein Molecular Weight Marker (20 μl)
  2. Sample 1 (HIC) from your group
  3. Sample 2 (HIC or Lysate) from your group
  4. Sample 3 (HIC) from your group
  5. Sample 4 (Lysate) from your group
  6. Mark 12 Protein Molecular Weight Marker (20 μl)
  7. Sample 1 (HIC) from another group
  8. Sample 2 (HIC or Lysate) from another group
  9. Sample 3 (HIC) from another group
  10. Sample 4 (Lysate) from another group

Native gel result

Here's a gel from 2/11/20:

IMG 3156e

GFP and BFP are visible under fluorescent light. This gel hasn't been stained yet, so other proteins aren't visible.

Reading on the 6B site:

Electrophoresis.

SDS-PAGE Method.

SDS-PAGE Sample Preparation and Assay.

Protein Gel Calculations. Also take a look at the Protein Assay Spreadsheet to see if your protein concentrations are similar to those measured by other groups. What will you do if they're not?

How to do lab calculations for some general background.

pGLO: SDS-PAGE.

Thursday, Feb 13

You have two separate experiments to work on today:

Finish your protein gel by destaining and photographing it. Start the destain process at the beginning of lab. Pour off the stain, rinse the gel briefly in deionized water, then leave the gel soaking in water while you do the PCR lab, and get the photo at the end. Remember that we have some denaturing gels and some non-denaturing (native) gels. If you have a non-denaturing gel, get photos with both uv light and visible light illumination.

Gel results

Two SDS-PAGE gels:

2X SDS PAGE

The SDS-PAGE gels mostly produced well-defined bands. By comparing the marker lanes to the experimental lanes, it was possible to see where the GFP band would be expected. I labeled two of the marker bands that are close to the size of GFP (GFP is around 28 kiloDaltons, or kDa). In these gels, GFP is clearly visible in samples 1 and 3, which are expected to have GFP, and less visible or absent in sampes 2 and 4, which shouldn't have GFP, assuming the gels were loaded according to the instructions above.

Lanes:

  1. Mark 12 Protein Molecular Weight Marker (20 μl)
  2. Sample 1 (HIC) from your group
  3. Sample 2 (HIC or Lysate) from your group
  4. Sample 3 (HIC) from your group
  5. Sample 4 (Lysate) from your group
  6. Mark 12 Protein Molecular Weight Marker (20 μl)
  7. Sample 1 (HIC) from another group
  8. Sample 2 (HIC or Lysate) from another group
  9. Sample 3 (HIC) from another group
  10. Sample 4 (Lysate) from another group

Native PAGE (one gel, before and after staining):

Native gel stain prestain

GFP and BFP (blue fluorescent protein) were clearly visible before staining the gel. Unfortunately, the fluorescence disappeared completely after staining. I don't know why, but I assume the staining solution denatured the protein. Unfortunately, the molecular weight marker (which is prepared in SDS running buffer) didn't show any clearly defined bands, so it's useless on the native gels. For this gel, I measured the location of the fluorescent bands on the pre-staining picture and then put an arrow in the corresponding location of the post-staining photo. I don't see any indication of the GFP being present in some lanes and absent in others. In this gel, it looks like the 8 lanes with bacterial samples were placed in the middle, with markers in lanes 1 and 10. All but one of the bacterial samples show fluorescence. It's possible that the gel could be flipped between the two pictures.

Conclusion: The native gels were great for showing fluorescence before staining, but didn't allow us to compare stained to fluorescent bands. Both types of gels allowed us to see GFP, in different ways.

PV92 PCR: prepare DNA templates and perform PCR reactions.

Tuesday, Feb 18

PV92 PCR: run DNA gel. For the small PCR products expected in this experiment, prepare your gel with 2% agarose.

You have 8 lanes on your gel, and 8 PCR samples. You should use a molecular weight marker, so you'll have to leave out one of your PCR samples. If you have 4 student PCR reactions, load your gel like this:

  1. Student PCR: 10 μl
  2. Student PCR: 10 μl
  3. Student PCR: 10 μl
  4. Student PCR: 10 μl
  5. Positive control PCR: 5 μl
  6. Positive control PCR: 5 μl
  7. Negative control PCR: 10 μl
  8. 100 bp DNA ladder (molecular weight marker): 10 μl

Mix each sample with sample buffer, using the parafilm technique. For the positive control PCRs, I recommend a smaller volume because these reactions often produce a large amount of DNA, which tends to smear the gel.

Results

Only about half the groups had good results on this. I think everyone had success with the positve and negative controls, meaning that the cocktail and the thermal cycling were good. The student DNA samples were less successful, though. The positive control templates start with a high concentration of DNA, so they are easy to amplify. If your samples didn't work and your positive controls did, it's probably due to either low quality or low quantity of template DNA in your sample. One possible problem is getting chelex from your template into the PCR reaction. Another possible problem is that you didn't have enough cells to begin with (although it shouldn't take much template DNA if the PCR is working well.)

Thursday, Feb 20

PTC PCR: Taste test and PCR reactions. If your PV92 PCR was successful, you can use the same DNA samples for this experiment. If your first PCR didn't work for a particular sample, you should prepare DNA from that person again.

Tuesday, Feb 25

PTC PCR: Restriction digests. Unlike the PV92 experiment, for this one you'll need to cut your PCR product with a restriction enzyme before you can tell the alleles apart.

Thursday, Feb 27

 PTC PCR: Electrophoresis.

Results of PTC PCR and restriction digest:

IMG 0043e

The PCR worked well for this group, producing bands in all the lanes except the negative control (I think). However, there are some problems. The main problem is that all the lanes seem to show the same heterozygous genotype. If both alleles are common in the population, then some individuals would be homozygous for one allele, some homozygous for the other. The fact that there are two bands in each lane might indicate that two alleles are present, or it might indicate that the restriction digest didn't cut all of the DNA. The best way we could find out whether the digest is working completely would be to have a positive control sample for which all the DNA is expected to be cut.

There may also be another problem with the results shown here. There should be two different positive controls: one heterozygous (two bands) and homozygous (only the upper, 303 bp band). I think the two positive controls look the same. It's possible that there could be cross-contamination from one sample to another.

Quiz 4!

Tuesday, Mar 3

 Bacteriophage.

Thursday, Mar 5

Bacteriophage plate results & PCR. Note that it will take an hour to prepare lysates for PCR, so get that started early.

Tuesday, Mar 10

 Phage PCR gel.

Here's a good result from the morning lab:

Phage PCR results

The PCR results are as expected. Each lane on the gel serves a purpose:

  1. Phage lysate: The plug lysate from a plate with plaques has phage DNA, and a PCR product was produced. If this sample produced no band, it could be because there was no phage DNA present, or because the PCR failed. We would need to look at the positive control to see if the PCR worked at all.
  2. No-phage lysate: The plug lysate from a plate with no phage and no plaques does not contain phage DNA, so no phage PCR product was produced. If there was a band here, it could mean that the PCR is copying some non-phage DNA that is present in the bacteria, or maybe the cocktail was contaminated with phage DNA.
  3. Positive control (T2 phage stock solution): Phage PCR product was produced as expected. Tests the PCR reaction with a known template, in case the phage lysate didn't show a product.
  4. Negative control (SM buffer alone): No product. If there was a product here, it would indicate that the cocktail was contaminated. If that was the case, the bands in the other lanes would be meaningless, because they could be due to contamination.

You should be able to explain the meaning of the results for the lab final.

Thursday, Mar 12

 Quiz & review

Tuesday, Mar 17

 Open review

Thursday, Mar 19

Lab Final

A- A A+