Lab 4a, 4b, 4i, 4j
Materials:
- Analytical Balance
- Tabletop Milligram Balance
- 7.6 x 7.6 cm Weigh Paper
- 3.5 x 3.5 Weigh Boat
- Lab Scoops
- Sodium Chloride
-15 mL Capped Tubes
- Tube Racks
- TRIS
- EDTA
- Disodium Salt
- 125 mL Bottle
- 100 mL Graduated Cylinder
- pH Paper
- Hydrochloric Acid
- Sodium Hydroxide
- Glass Rods
- 50 mL Beakers
- Salmon Sperm DNA
- 2 mL Pipette
- P-1000 Micropipette, and tips
- 95% Ethanol
- Permanent Marker Pens
- 1L Tripour Plastic Peaker
- 40X TAE Buffer Concentrate
- 600 mL Beakers
- Agarose
- 250 mL MEdia Bottle
- Microwave Oven
- Hot Hands Protector
- Horizontal Gel Box
- 65 degree Celcius water bath
Lab 4a and 4b
Precipitation - taking something out of a solution
DNases - breaks down DNA
NaCL - positive charges of Na+ bind DNA so it can clump together and form a precipitate
Tris - maintains pH (7-8)
EDTA - prevents DNases activity
Purpose:
make 10ml of 5M NaCL
make 100ml of 10mM Tris, 1mM EDTA
M = 1mole/1L
mM = 1/1000 mole/L
1 mole = 6.02 x 10^23 particles
5M = (5)(6.02 x 10^23 partles NaClL)
Molarity Calculations
(Molarity)(Volume)(Formula Weight) = g substance needed
Solution 1: 10ml of 5M NaCl
Formula: (M)(V)(FW)
(5M)(0.01L)(58.44g/mol) = 2.92gNaCl NaCl formula weight = 58.44g/mol
Solution 2: TE
Tris (0.01M/L)(0.1L)(157.6g/mol) = 0.158gTris analytical balance
EDTA (0.001M/L)(0.1L)(372.24g/mol) = 0.037gEDTA analytical balance
Label
what it is - NaCl
concentration - 5M
date 10/9/14
initials ZM
period - 2/3
designed 7.5 - 8.5pH
pH around 6.3 - 6.5 need to raise pH
add base NaCl
final pH - 7.7
C1(stock solution concentration)V1(volume of stock solution to use) = C2(final desired concentraion)V2(final desired volume)
C1 = 4mg/ml
V1 = ?
C2 = 2mg/ml
V2 = 2ml
Purpose:
1. Dilute DNA with TE in beaker, observe - 1ml DNA, 1ml TE
2. add NaCl
3. Add 4ml EtOH (trickle down side) Observe
4. Spool DNA
5. Put DNA into new tube with 2ml fresh TE and label
Observations:
1. The substance looked transparent and somewhat jelly like
2. The substance was still transparent with an additional layer, DNA began to show
3. DNA was dense and had more of a white color than the rest of the substance
4. DNA seemed clumpy
Pouring agarose gels - Lab 4i
-8% agarose in 1x TAE (tris-acetate-edta)
-TRIS buffer
-CH3COO (acetate) - prevents DNA clumping
- EDTA - prevents DNases
1 X TAE
Make from 40x stock
C1V1 = C2V2
V1 = C2V2/C1
C1 = Stock concentration
V1 = ?
C2 = final concentration
V2 = final volume
V1 = (1)(500)/400 = 12.5ml 40xTAE
+ H20 up to 500 ml (qs to 500ml)
50ml 0.8% agarose in 1x TAE
% by volume
0.8% of 50ml
0.008% x 50 = 0.4g agarose
in 50ml
1 x TAE
In erlenmeyer flask
Procedure:
1. Make 500ml 1xTAE + 50ml .8% agarose
Use 500ml g.c + 250ml erlenmeyer flask + 25 ml pipet/red filler
a. Add agarose to 100ml 1xTAE in flask
b. Heat to boil and dissolve -
heat - swirl - heat - swirl until clear
c. Let cool until you can touch flask for few seconds
d.Pour in prepared mold and let cool
Lab 4j
Procedure:
1. Remove tape from gel, place in gel tank
2. Pour TAE over gel until covered , gently remove combs
3. Prepare samples
- 20ml DNA + 4ml 6x loading dye
- Spin 2 sec. in mini centrifuge
4. Load samples onto gel
5. Put corer on gel tank, plug in to power supply
6. Run at 110v for about 45 minutes
7. Stain several hours with EtBr (Ethidum Bromide) rinse and observe with uv light
Load dye -
- dyes to track gel progress (runs in front of samples)
- Glycerol - to make samples sink into well
- Analytical Balance
- Tabletop Milligram Balance
- 7.6 x 7.6 cm Weigh Paper
- 3.5 x 3.5 Weigh Boat
- Lab Scoops
- Sodium Chloride
-15 mL Capped Tubes
- Tube Racks
- TRIS
- EDTA
- Disodium Salt
- 125 mL Bottle
- 100 mL Graduated Cylinder
- pH Paper
- Hydrochloric Acid
- Sodium Hydroxide
- Glass Rods
- 50 mL Beakers
- Salmon Sperm DNA
- 2 mL Pipette
- P-1000 Micropipette, and tips
- 95% Ethanol
- Permanent Marker Pens
- 1L Tripour Plastic Peaker
- 40X TAE Buffer Concentrate
- 600 mL Beakers
- Agarose
- 250 mL MEdia Bottle
- Microwave Oven
- Hot Hands Protector
- Horizontal Gel Box
- 65 degree Celcius water bath
Lab 4a and 4b
Precipitation - taking something out of a solution
DNases - breaks down DNA
NaCL - positive charges of Na+ bind DNA so it can clump together and form a precipitate
Tris - maintains pH (7-8)
EDTA - prevents DNases activity
Purpose:
make 10ml of 5M NaCL
make 100ml of 10mM Tris, 1mM EDTA
M = 1mole/1L
mM = 1/1000 mole/L
1 mole = 6.02 x 10^23 particles
5M = (5)(6.02 x 10^23 partles NaClL)
Molarity Calculations
(Molarity)(Volume)(Formula Weight) = g substance needed
Solution 1: 10ml of 5M NaCl
Formula: (M)(V)(FW)
(5M)(0.01L)(58.44g/mol) = 2.92gNaCl NaCl formula weight = 58.44g/mol
Solution 2: TE
Tris (0.01M/L)(0.1L)(157.6g/mol) = 0.158gTris analytical balance
EDTA (0.001M/L)(0.1L)(372.24g/mol) = 0.037gEDTA analytical balance
Label
what it is - NaCl
concentration - 5M
date 10/9/14
initials ZM
period - 2/3
designed 7.5 - 8.5pH
pH around 6.3 - 6.5 need to raise pH
add base NaCl
final pH - 7.7
C1(stock solution concentration)V1(volume of stock solution to use) = C2(final desired concentraion)V2(final desired volume)
C1 = 4mg/ml
V1 = ?
C2 = 2mg/ml
V2 = 2ml
Purpose:
1. Dilute DNA with TE in beaker, observe - 1ml DNA, 1ml TE
2. add NaCl
3. Add 4ml EtOH (trickle down side) Observe
4. Spool DNA
5. Put DNA into new tube with 2ml fresh TE and label
Observations:
1. The substance looked transparent and somewhat jelly like
2. The substance was still transparent with an additional layer, DNA began to show
3. DNA was dense and had more of a white color than the rest of the substance
4. DNA seemed clumpy
Pouring agarose gels - Lab 4i
-8% agarose in 1x TAE (tris-acetate-edta)
-TRIS buffer
-CH3COO (acetate) - prevents DNA clumping
- EDTA - prevents DNases
1 X TAE
Make from 40x stock
C1V1 = C2V2
V1 = C2V2/C1
C1 = Stock concentration
V1 = ?
C2 = final concentration
V2 = final volume
V1 = (1)(500)/400 = 12.5ml 40xTAE
+ H20 up to 500 ml (qs to 500ml)
50ml 0.8% agarose in 1x TAE
% by volume
0.8% of 50ml
0.008% x 50 = 0.4g agarose
in 50ml
1 x TAE
In erlenmeyer flask
Procedure:
1. Make 500ml 1xTAE + 50ml .8% agarose
Use 500ml g.c + 250ml erlenmeyer flask + 25 ml pipet/red filler
a. Add agarose to 100ml 1xTAE in flask
b. Heat to boil and dissolve -
heat - swirl - heat - swirl until clear
c. Let cool until you can touch flask for few seconds
d.Pour in prepared mold and let cool
Lab 4j
Procedure:
1. Remove tape from gel, place in gel tank
2. Pour TAE over gel until covered , gently remove combs
3. Prepare samples
- 20ml DNA + 4ml 6x loading dye
- Spin 2 sec. in mini centrifuge
4. Load samples onto gel
5. Put corer on gel tank, plug in to power supply
6. Run at 110v for about 45 minutes
7. Stain several hours with EtBr (Ethidum Bromide) rinse and observe with uv light
Load dye -
- dyes to track gel progress (runs in front of samples)
- Glycerol - to make samples sink into well
Conclusion
When we did this experiment it didn't go as planned. There was no movement of the DNA when we casted the light on it because:
The solution didn't respond
The staining time was too short
The solution was made incorrectly
DNA ran off the gel
We could do different tests that could help us figure out what went wrong.
1. Dot blot test with CtBr solution and put DNA on it
2. Make EtBr again and re-stain gels.
Reflection
We did not achieve the end goal, but my group and I worked well together. We could have been more efficient if everybody worked on different things instead of all working on one thing at a time, but we still finished. I enjoyed pipetting a lot and thought it was entertaining and quite interesting. I think making solutions and adjusting pH are good because we make sure we have the right amount by being extremely careful. We never cross-contaminated the solutions which could cause the experiment to have false results. This was a very interesting lab because even though the lab didn't work we still had an opportunity to learn about new things and work with new equipment that we never had the chance to work with before.
When we did this experiment it didn't go as planned. There was no movement of the DNA when we casted the light on it because:
The solution didn't respond
The staining time was too short
The solution was made incorrectly
DNA ran off the gel
We could do different tests that could help us figure out what went wrong.
1. Dot blot test with CtBr solution and put DNA on it
2. Make EtBr again and re-stain gels.
Reflection
We did not achieve the end goal, but my group and I worked well together. We could have been more efficient if everybody worked on different things instead of all working on one thing at a time, but we still finished. I enjoyed pipetting a lot and thought it was entertaining and quite interesting. I think making solutions and adjusting pH are good because we make sure we have the right amount by being extremely careful. We never cross-contaminated the solutions which could cause the experiment to have false results. This was a very interesting lab because even though the lab didn't work we still had an opportunity to learn about new things and work with new equipment that we never had the chance to work with before.