Our group was amazing! It included me, Sierra, and Tyler. We worked really hard to focus and learn something about the pGLO gene(also known as jellyfish gene!!) By the way, jellyfish hurt, a lot!!
The PROCESS!!
1. label one closed test tube +pGLO and another -pGLO. Place in the foamy tubey thingy..
2. open tubes and use pipet to transfer 250ul of the CaCl2. Place on ice.
4. Use loop to pick up a single colony of bacteria from the starter plate. Get the +pGLO tube and immerse the loop into the transformation solution at the bottom of the tube. Spin the loop "washing machine" until the entire colony is dispersed. Place the tube back in the ice. Repeat with -pGLO with a new sterile loop.
5. Examine plasmid DNA with the UV lamp. Immerse new into plasmid DNA stocktube. Withdraw a loopful. Should be a film of plasmid across the ring, like you're about to blow a bubble. Mix loopful into the cell suspension of the +pGLO tube. Return it to the ice. Don't add plasmid DNA to the -pGLO tube.
6. Incubate tubes on ice for 10 minutes. Bottom of tubes should make contact with the ice.
7.Label four agar plates on the bottom as follows: one LB/amp plate label +pGLO; LB/amp/ara plate label -pGLO; LB/amp label -pGLO; and the LB plate -pGLO.
8. HEAT SHOCK!! Transfer both tubes into the water bath that's set at 42 degrees Celsius for exactly 50 seconds. (Push the tubes all the way down in the rack so the bottom of the tubes make contact with the water. Replace both tubes on ice immediately. Incubate in ice for 2 minutes.
9. Take off ice and open a tube. Use a new pipet to add 250 ul of LB nutrient broth to the tube and reclose it. Repeat with a new pipet to the other tube. Incubate at room temp for 10 minutes.
10. Tap closed tubes with finger to mix(or flick it). Use new sterile pipet for each tube, pipet 100ul of the transformationa nd control suspensions to appropriate plates.
11. Spread suspensions evenly with a sterile loop.
12. Stack of plates and tape them together, upside down. Put it in the Biology rooms' incubator overnight which is set at 37 degrees Celsius.
Review Answers:
Observations:
+pGLO; LB/amp: has the gene but it isn't "turned on". It has a medium to heavy population which is white and yellow. Our estimated count was 130. Transformation plates
+pGLO; LB/amp/ara: has the protein that is turned on, which makes it glow. It has a medium to heavy population that's yellow then a light white. Our estimated count is 149. Transformation plates
-pGLO; LB/amp: has no population and no bacteria!! Control Plates
-pGLO; LB: has a HEAVY bacterial lawn!!! Control Plates
Analysis:
Plasmid doesn't make a difference to the health.
Our goal is to see if it glows or doesn't glow
The amp kills unless a gene from the plasmid is LB/amp +/-
Definitions:
amp = antibiotic
ara = arabinose sugar (activates some genes)
The PROCESS!!
1. label one closed test tube +pGLO and another -pGLO. Place in the foamy tubey thingy..
2. open tubes and use pipet to transfer 250ul of the CaCl2. Place on ice.
4. Use loop to pick up a single colony of bacteria from the starter plate. Get the +pGLO tube and immerse the loop into the transformation solution at the bottom of the tube. Spin the loop "washing machine" until the entire colony is dispersed. Place the tube back in the ice. Repeat with -pGLO with a new sterile loop.
5. Examine plasmid DNA with the UV lamp. Immerse new into plasmid DNA stocktube. Withdraw a loopful. Should be a film of plasmid across the ring, like you're about to blow a bubble. Mix loopful into the cell suspension of the +pGLO tube. Return it to the ice. Don't add plasmid DNA to the -pGLO tube.
6. Incubate tubes on ice for 10 minutes. Bottom of tubes should make contact with the ice.
7.Label four agar plates on the bottom as follows: one LB/amp plate label +pGLO; LB/amp/ara plate label -pGLO; LB/amp label -pGLO; and the LB plate -pGLO.
8. HEAT SHOCK!! Transfer both tubes into the water bath that's set at 42 degrees Celsius for exactly 50 seconds. (Push the tubes all the way down in the rack so the bottom of the tubes make contact with the water. Replace both tubes on ice immediately. Incubate in ice for 2 minutes.
9. Take off ice and open a tube. Use a new pipet to add 250 ul of LB nutrient broth to the tube and reclose it. Repeat with a new pipet to the other tube. Incubate at room temp for 10 minutes.
10. Tap closed tubes with finger to mix(or flick it). Use new sterile pipet for each tube, pipet 100ul of the transformationa nd control suspensions to appropriate plates.
11. Spread suspensions evenly with a sterile loop.
12. Stack of plates and tape them together, upside down. Put it in the Biology rooms' incubator overnight which is set at 37 degrees Celsius.
Review Answers:
- I would expect to find bacteria on the LB plate because I never saw any plasmid.
- Genetically transformed bacterial cells would be located on the LB/amp/ara and LB+ plate. Because the antibiotic is selecting.
- LB/amp plates should be compared to determine any genetic transformation because it can be used as a control.
- The controls were: same bacteria, same temperature, and the same amount of bacteria. In this case the LB was the control.
Observations:
+pGLO; LB/amp: has the gene but it isn't "turned on". It has a medium to heavy population which is white and yellow. Our estimated count was 130. Transformation plates
+pGLO; LB/amp/ara: has the protein that is turned on, which makes it glow. It has a medium to heavy population that's yellow then a light white. Our estimated count is 149. Transformation plates
-pGLO; LB/amp: has no population and no bacteria!! Control Plates
-pGLO; LB: has a HEAVY bacterial lawn!!! Control Plates
Analysis:
Plasmid doesn't make a difference to the health.
Our goal is to see if it glows or doesn't glow
The amp kills unless a gene from the plasmid is LB/amp +/-
Definitions:
amp = antibiotic
ara = arabinose sugar (activates some genes)
Hello,
ReplyDeleteThey can be classified into several categories depending on their functions. Let's examine some of the types of pipettes used in laboratories.
Micropipette