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Troubleshooting Amino Labs Wetware Kits
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STEP 1: Making LB agar plates
When I place the plates 4 inches over a picture or writing, it was blurry. The LB agar plates were also soft and easy to puncture with the loop.
When I placed the plate 4 inches over a picture or writing, I could see it clearly. The LB agar plates were very transparent and the agar solidified nicely
The agar was soft and opaque and I didn’t get colonies on the surface. I punctured the surface of the agar with the loop.
The agar was transparent and solid. The cells streaked, and after incubation cells grew and I got colonies
The agar was transparent and solid and I streaked the cells, but after incubation I got solid lines of bacteria (lawns), but I did not get any colonies
STEP 2: Steaking & Growing Blank Cells
The agar was transparent and solid and I streaked the cells, but after incubation the cells did not grow.
Rolling Boil
Before you add the LB agar powder, ensure that your sterile water has a rolling boil
Back to the Beginning
Foaming/Boiling Molten LB Agar
After you add the LB agar powder and swirl to mix in, microwave for further 4 second intervals until you see further boiling. You will see a foam rising above.
After you see this, give another swirl to mix, and continue on to the next step, adding selection.
Diagnosis: Insufficient Boiling
Practice with Jell-O
Practice tip: making Jell-O follows similar principles as making LB agar plates. You can practice by buying a pack of Jell-O and making it.
Also, Jell-O is delicious!
OK, I’ll try this next time!
LB agar plates that are soft makes it very difficult to both streak blank cells on the surface to get fast growing colonies, and to incubate your plates. Here are some tips to ensure perfect LB agar plates.
The “+” control did grow
The “+” control did not grow
I’m pretty sure I dipped it in
OK, I’ll try “+” cells
When working with microbes, they are so small you can’t see them with your eyes! When you dipped your yellow loop into the tube of cells, you may not have touched the surface of the agar or inserted it into the agar.
Next time, you can double check by making sure the loop is wet from contacting the blank cells and agar in the tube.
If you haven’t already, try this by completing the “+ control” exercise that is normally done on Day 2.
Diagnosis: Cells on yellow loop?
You found the problem! When contacting us about the Success Guarantee, mention that it seems your LB agar was not completely boiled and this led to your agar being soft and opaque.
Also, be sure to send a picture of your four petri dishes, and if possible the “softness” of the agar! No worries if your plates were soft and you didn’t complete the positive control or the rest of the streaking exercises.
Lastly, as part of the learning process, include a short description, from your perspective, of the moment when you think the experiment went wrong. You can also tell us how to improve Virtual Bioengineer to help make sure no one else encounters this situation.
Email for Success Guarantee
Got it
Tips on making LB agar plates
Congrats on getting some growth, this is a big first step, however, it is important to have lots of single colonies that are growing fast.
Soft agar makes it difficult to get well streaked plates and also to collect the bacteria that are growing!
Because your "+" cells grew the cells must be viable. This may indicate that you may not have fully dipped the yellow loop in the the blank cells in the tube containing the stab of cells. Alternatively, might you have accidentally used a selective plate when streaking your blank cells? If so, click accordingly below.
When contacting us about the Success Guarantee, mention that it seems you may not have fully dipped your yellow loop fully into the tube of blank cells.
Also, be sure to send a picture of your four petri dishes, and we recommend that if you haven’t already, streaking the “+ cells” on your selective “+” plate. This is normally part of Day 2 activities.
Lastly, as part of the learning process, include a short description, from your perspective, of the moment when you think the experiment went wrong. Also, tell us whether your positive control worked.
Whoops, I used a selective plate
I got one colony of engineered bacteria, and my positive control cells grew
I got no engineered colonies, but my positive control cells grew
(+)
STEP 3: Getting Engineered Colonies
I got no engineered colonies and I got no growth on my “+” plate
(E)
I got no engineered colonies but there was a smear of growth across the entire plate and the “+” cells grew but didn’t turn colourful
After plating my heat shocked and recovered cells on the “E” plate:
Diagnosis: Plate, Temperature, Time
OK, I’ll incubate longer
Oops, I used a selective plate
Temp is ~ 37 °C (16-24 hours) or Time is greater than 72 hours, and I used a non-selective plate
Blank cells: Is it possible that you accidentally streaked blank cells onto a selective plate? Antibiotics in a selective plate would not allow the blank cells to grow
All cells: If you do not have a DNA Playground or an incubator, it is more difficult to reach and maintain 37 °C. If you are incubating your cells at lower than 37 °C it can take longer for the cells to grow, even up to four days to see colonies! Perhaps wait a little longer to see if your cells grow.
If you think you dipped the yellow loop into the cells, there are a couple possibilities for not seeing growth of colonies.
-collect small colonies? (not the lawn bacteria)
-fill the centre of the loop with cells?
-ensure the T. Buffer was at the bottom of the tube? (should be about 5 mm of liquid)
DNA
After adding cells to the T. Buffer and mixing, when you added your DNA, did you double check to make sure that there was DNA liquid inside of the blue loop hole?
Temperature
Keeping the cells cold while they are in the T. buffer, and while you’re adding and mixing in the DNA is important. If they warm up, or if you held the tube with your fingers while mixing, your fingers can warm up the tube and kill cells at a much faster rate than if they are kept cold.
I did these all correctly
There are several key factors that determine the success of the transformation step. These include cells, DNA, and temperature. Because your + cells worked, your plates must have been well made and your cells were viable:
Diagnosis: Cells, DNA, Temperature
Cells
When collecting your fast growing colonies, did you:
The only other possibility for not seeing colonies might involve that you do not have a DNA Playground or an incubator and your DIY incubator setup isn’t quite getting to 37 °C. This could mean that:
1) your blank cells weren’t growing so fast due to the lower temperature, which causes their membranes to be more rigid and DNA isn’t able to get across the membrane so easily.
2) you are incubating your transformed cells at lower than 37 °C means they can take up to 4 days to become visible. You may want to incubate a bit longer and see if colonies appear. In future experiments it is important to have an incubator or environment that is 37 °C.
If you completed all of the above steps properly and your positive control cells grew, then you should have had at least one colony! If this is your first time, you may have just missed something and we urge you to try again! Please contact us for the Success Guarantee, and we’ll be happy to send you another kit.
I didn’t do the “+” cells yet - I’ll try that
If your LB agar plates were made right and both your blank cells and your “+” cells did not grow, this could mean that the cells have died.
Typically the cells will be viable for 6 months from delivery - check the expiry date on the tube of cells. However, the shelf life of the cells can be shortened if:
The cells were not stored in the fridge. Keeping your cells in the fridge slows their growth and metabolism and puts them into “suspended animation”. This keeps them viable for the 6 month time period
If the cells froze. Freezing cells will kill them. As water crystals form inside the cells, they break the cell membrane and kill the cells
If the cells were heated to more than 50 °C for sustained periods during any point of their delivery or storage. While E. coli cells can survive at up to 50 °C for periods of time, the cells will begin to die.
You may have found out why the exercise could have gone wrong! After you’ve tried this suggestion, check back with this troubleshooter.
Remember, troubleshooting experiments is a critical part of doing genetic engineering and scientific reserach.
If your (E) plate didn’t have colonies of engineered bacteria growing, but did have a film or smear across the entire plate where it looks like bacteria grew, then the likely cause is the antibiotic concentration in the LB agar is too low or non-existent.
This is further reinforced in your positive control. The positive control cells will grow on a non-selective plate, however, they will not change color very well because there is no selective pressure.
A possible cause of this outcome is you may have forgotten to put the antibiotic pill into the molten LB agar after creating your non-selective plate, or, you may not have fully dissolved the capsule so that the antibiotics were fully mixed into the agar.
This is an honest mistake!
When contacting us about the Success Guarantee, mention that you accidentally streaked your blank stab cells onto a plate with antibiotics and they didn’t grow.
Also, be sure to send a picture of your four petri dishes, and we recommend that if you haven’t already, streaking the “+ cells” on your selective “+” plate. This is normally part of Day 2 activities.
Lastly, as part of the learning process, include a short description, from your perspective, of the moment when you think the experiment went wrong. It would be great if you could recommend on how to make Virtual Bioengineer better so nobody else encounters this situation!
Congratulations on getting some blank cells to grow! If you did not get any blank cell colonies, this could be due to:
1) during each streaking event, did you use a new loop each time? Or did you use one loop to complete all three lines? If you use a single loop, this would cause lots of bacteria to be deposited on each zig zag line. This would result in you not getting much less bacteria on zig zag lines 2 and 3. Because of this you may not see individual bacteria, but rather solid lines of bacteria.
2) during the streaking events, did you only dip the first loop into the stab agar? Or did you dip each new loop into the stab agar before making the zig zag? If this is the case, you’re only supposed to dip into the stab agar with the first loop to collect lots of cells. After that you should use new clean loops to complete the rest of the zig zags. This is so that you can get less and less cells on zig zag lines 2 and 3 - less and less cell means you will ultimately get single bacteria that will grow into colonies of fast growing bacteria.
Using colonies of fast growing bacteria substantially increase your chances of sucessfully transforming your blank cells. Continue with the exercise as you may still be successful. If you’re not, the fact that you don’t have fast growing colonies may be the reason why your transformation did not work.
OK, I’ll keep trying with lawn bacteria
Congratulations!!!
Getting a single engineered colony is the difference between not being a genetic engineer and being a genetic engineer! It is very common to get one colony of engineered bacteria the first or even second time you try the Engineer-it Kit experience. As you continue to practice you will likely get 5x the number of colonies next time, and even another 5x the time after that!