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Project Introduction
Preparation of the DNA sample for CIDEB-UANIL’s team in Mexico, Tuesday 3rd June 2014.
There was no pre-prepared sample of the DNA that the team wanted; however, there were 3 different samples of glycerol stock of bacteria which contained it. It is against regulations to send bacteria internationally, so we had to remove the DNA from the bacteria to send them their sample.
Firstly, we removed the glycerol stock from the freezer so allow it to defrost. Whilst waiting for that to occur, we set our agar plates and tubes of LB medium in which to grow the bacteria. We put the jar of agar into the microwave to melt it down, and whilst that was melting we poured 10 millilitres of LB medium (a liquid containing different nutrients to aid the bacteria’s growth) into 3 different tubes. We did the agar plates in addition to putting the glycerol stock straight into the LB as it doesn’t always like to grow straight away in the LB. Additionally, we can get a purer sample as we can separate colonies.
We poured out around 10 millilitres of the agar jelly into 6 different plates; each glycerol stock sample would get 2 different plates. Whilst we waited for them to set, we put 10 micro litres of each of the 3 glycerol stock samples into their respective tubes of LB. We put the tubes of LB medium into a shaker at 250 rotations per minute, so that the bacteria was always thoroughly mixed in the medium and could get to all of the nutrients.
When the agar had set, we used streakers to spread some of the glycerol stock onto the jelly. One was used to put the first 3 lines, a clean one used to spread that to get the sample as thin as possible. This is to make the sample small enough for only one colony to grow in a particular spot, which means when we grow that one colony in the LB medium the DNA will all be clones of each other.
The agar plates were placed in an incubator at 37 degrees Celsius, upside down to ensure that no condensation could drip onto the samples and possibly contaminate them.
We left both the tubes of LB medium and the agar plates overnight to grow the bacteria.
Wednesday 4th June, 2014.
After around 22 hours we took the plates out of the incubator and the tubes from the shaker. Only 2 of the 3 tubes of LB medium had grown; the liquid had gone cloudy.
We put those 2 tubes of LB medium into the large centrifuge to make the bacteria into pellets and be able to tip off the medium. We taped the two tubes of bacteria together and put them in the freezer. The reason for tipping the medium off before freezing the bacteria is to ensure that there is no liquid that can form ice crystals, which could in turn break the cells open. We froze the pellets of bacterial cells quickly to preserve them and not allow the concentration of cells to decrease. Bacteria grow exponentially until they plateau, soon after which they start to die and be degraded, thus lowering the concentration of bacterial cells.
All but one of the samples in the agar jelly grew; the 5 which had grown had little white spots on them – each spot was a colony of bacteria. The empty plate had not grown anything.
We chose the 4 best plates with the most growth on them; ensuring that there was at least 1 colony from each sample of the glycerol stock. We put 10 more millilitres of LB medium into 4 new tubes, using new streakers to scrape off 1 colony of bacteria and put it into a tube of medium. These tubes were put into the same shaker, again at 250 spins per minute, and left overnight.
Thursday 5th June, 2014.
After removing the tubes from the shaker again around 22 hours later, we found all of them had grown. We put them into the large centrifuge to make the pellets and again tipped off the medium once they were done.
With these pellets we completed a mini-prep with the following steps:
1. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a
microcentrifuge tube.
2. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube
4–6 times.
3. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube
4–6 times.
4. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top
microcentrifuge.
5. Apply the supernatant from step 5 to the QIAprep spin column by decanting or
pipetting.
6. Centrifuge for 30–60 s and discard the flow-through.
7. Wash the QIAprep spin column by adding 0.5 ml Buffer PB.
8. Wash the QIAprep spin column by adding 0.75 ml Buffer PE.
9. Centrifuge for 30–60 s and discard the flow-through
10. Centrifuge for 1 min to remove residual wash buffer
11. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube.
12. Add 50 μl Buffer EB or water to the QIAprep spin column, stand for 1 min, and centrifuge for 1 min.
We measured the end concentration of the DNA samples using the nanodrop, and then sent off all 4 samples with DHL.
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Growing the bacteria needed for our own project, Tuesday 10th June, 2014.
We had 5 different glycerol stock samples of Ecoli bacteria to grow for our own project. One of them was simply the Ecoli with no specific plasmids in it. Another 3 of them contained plasmids with IRRE (a promoter and RBS), and the last one contained plasmids with indoli. To begin, we removed the glycerol stock from the freezer to allow it to defrost. Whilst waiting for that to occur, we set our agar plates and tubes of LB medium in which to grow the bacteria. We put the jar of agar into the microwave to melt it down, and whilst that was melting we poured 10 millilitres of LB medium – a liquid containing different nutrients to aid the bacteria’s growth - into 11 different tubes; 2 per glycerol stock sample and a control to make sure there was no contamination. We did the agar plates in addition to putting the glycerol stock straight into the LB as it doesn’t always like to grow straight away in the medium.
We poured out around 10 millilitres of the agar jelly into 10 different plates; each glycerol stock sample would get 2 different plates. Whilst we waited for them to set, we put 10 micro litres of each of the 3 glycerol stock samples into their respective tubes of LB. We put the tubes of LB medium into a shaker at 250 rotations per minute, so that the bacteria was always thoroughly mixed in the medium and could get to all of the nutrients.
When the agar had set, we used streakers to spread some of the glycerol stock onto the jelly. One was used to put the first 3 lines, a clean one used to spread that to get the sample as thin as possible. This is to make the sample small enough for only one colony to grow in a particular spot, which means when we grow that one colony in the LB medium the DNA will all be clones of each other.
The agar plates were placed in an incubator at 37 degrees Celsius, upside down to ensure that no condensation could drip onto the samples and possibly contaminate them.
We left both the tubes of LB medium and the agar plates overnight to grow the bacteria.
Wednesday 11th June, 2014.
All the LB tubes grew overnight in the shaker, so we put them into the large centrifuge to make the pellets and tipped off the medium once they were done.
With these pellets we completed a mini-prep with the following steps:
1. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a
microcentrifuge tube.
2. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube
4–6 times.
3. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube
4–6 times.
4. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top
microcentrifuge.
5. Apply the supernatant from step 5 to the QIAprep spin column by decanting or
pipetting.
6. Centrifuge for 30–60 s and discard the flow-through.
7. Wash the QIAprep spin column by adding 0.5 ml Buffer PB.
8. Wash the QIAprep spin column by adding 0.75 ml Buffer PE.
9. Centrifuge for 30–60 s and discard the flow-through
10. Centrifuge for 1 min to remove residual wash buffer
11. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube.
12. Add 50 μl Buffer EB or water to the QIAprep spin column, stand for 1 min, and centrifuge for 1 min.
When we completed the mini-prep we measured the concentrations of DNA with the nanodrop. For PCR you need a minimum of around 0.5ng/ul, and our concentrations fell below that. PCR wouldn’t have been effective on these samples, so we discarded them and set more LB medium tubes. We picked selected colonies off some of the agar plates we set along with our previous tubes and put them in the LB medium. We then placed them in the shaker once again overnight, hoping that the next day we would collect them and get high enough concentrations.
Thursday 12th June, 2014.
When we collected our LB tubes from the shaker the next day, we found that they had all grown. We used the same steps as before in order to carry out a further mini-prep. Thankfully the concentrations were just about high enough to be effectively used in PCR..
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Results
- Result 1 - Lorem ipsum ad his scripta blandit partiendo, eum fastidii accumsan euripidis in, eum liber hendrerit an.
- Result 2 - Lorem ipsum ad his scripta blandit partiendo, eum fastidii accumsan euripidis in, eum liber hendrerit an.
- Result 3 - Lorem ipsum ad his scripta blandit partiendo, eum fastidii accumsan euripidis in, eum liber hendrerit an.
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