Lab 4a, 4b, 4i, 4j-DNA
Materials for Lab 4
Analytical balance
Tabletop milligram balance
7.6 cm x 7.6 cm weigh paper
3.5 inches x 3.5 inches weigh boat
Lab scoops
Sodium chloride
Tubes, 15 mL, capped
Tube racks for 15 mL tubes
TRIS
EDTA, disodium salt
Bottle, 125 mL
Graduated cylinder, 100 mL
Ph paper
Hydrochloric acid
Sodium hydroxide
Glass rods
50 mL beakers
Salmon sperm sample
2mL pipet
Micropipet, P-1000
95% Ethanol
Sharpie
Plastic beaker 1L tripour
40x TAE buffer concentrate
600 mL beaker
Agarose
250 mL Media bottle
Microwave Oven
Hot hands protector
Horizontal gel box
65 degree Celsius water bath
Prepared agarose gel
Tube rack for 1.7 mL tubes
Reaction tubes, 1.7 mL
DNA samples
Yeast DNA 50 micrometers (g)/micrometer(liter) and loading dye
pBR322 50 micrometers (g)/micrometer(liter) and dye
Lambda 50 micrometers (g)/micrometer(liter) and loading dye
Other loading samples and dye
Gel loading dye 10X or 6X
Micropipet, P-10
Micropipet, P-100
Micropipet tips for P-100
Microcentrifuge
Lambda/ HimIII, 50 micrometers (g)/micrometer(liter) and dye
power supply
Ethidium bromide, 0.5 micrometer(g)/ mL
Gel photo imaging system
Thermal paper
Thermal paper
Large gloves
Safety plastic glasses
5.5 inches x 5.5 inches weigh boat
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
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
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 was clear and jelly like
2. The substance was clear with an added layer, DNA was appearing
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
I really enjoyed all of these projects and it was really fun being able to test the Dna in a real world application.My group worked very well and i would enjoy working with them in the future. The Dna gels were probably the most fun and at first it didn't work but we remade the dye and it worked so it was pretty fun and educational.
Materials for Lab 4
Analytical balance
Tabletop milligram balance
7.6 cm x 7.6 cm weigh paper
3.5 inches x 3.5 inches weigh boat
Lab scoops
Sodium chloride
Tubes, 15 mL, capped
Tube racks for 15 mL tubes
TRIS
EDTA, disodium salt
Bottle, 125 mL
Graduated cylinder, 100 mL
Ph paper
Hydrochloric acid
Sodium hydroxide
Glass rods
50 mL beakers
Salmon sperm sample
2mL pipet
Micropipet, P-1000
95% Ethanol
Sharpie
Plastic beaker 1L tripour
40x TAE buffer concentrate
600 mL beaker
Agarose
250 mL Media bottle
Microwave Oven
Hot hands protector
Horizontal gel box
65 degree Celsius water bath
Prepared agarose gel
Tube rack for 1.7 mL tubes
Reaction tubes, 1.7 mL
DNA samples
Yeast DNA 50 micrometers (g)/micrometer(liter) and loading dye
pBR322 50 micrometers (g)/micrometer(liter) and dye
Lambda 50 micrometers (g)/micrometer(liter) and loading dye
Other loading samples and dye
Gel loading dye 10X or 6X
Micropipet, P-10
Micropipet, P-100
Micropipet tips for P-100
Microcentrifuge
Lambda/ HimIII, 50 micrometers (g)/micrometer(liter) and dye
power supply
Ethidium bromide, 0.5 micrometer(g)/ mL
Gel photo imaging system
Thermal paper
Thermal paper
Large gloves
Safety plastic glasses
5.5 inches x 5.5 inches weigh boat
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
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
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 was clear and jelly like
2. The substance was clear with an added layer, DNA was appearing
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
I really enjoyed all of these projects and it was really fun being able to test the Dna in a real world application.My group worked very well and i would enjoy working with them in the future. The Dna gels were probably the most fun and at first it didn't work but we remade the dye and it worked so it was pretty fun and educational.