BCHN 213

Revision for practical exam

1

12 April 2023
 Good Laboratory Practice (GLP)
• GLP video is on efundi
• GLP starts on pg 76 of English study guide
• Conversions (pg 80) – NB!
• Calculations – NB!
• Serial dilutions
 Good Laboratory Practice (GLP)
• Buffers calculations
• You need to know you conversions!

C1V1 = C2V2 CiVi = CfVf

(__ μg/mL) (____L) = (___mg/L)(___μL) (__ μg/mL) (____L) = (___mg/L)(___μL)
C1 = Concentration that you start with (Stock solution)  Ci = initial concentration
V1 = Volume needed of Stock solution  Vi = initial volume
C2 = Concentration that you want  Cf = Final concentration
V2 = Volume needed to get to the concentration that you want  Vf = Final volume

• Conversions (pg 80) – NB!

 Good Laboratory Practice (GLP)
• You are supplied with the following:
1.   NaCl (Mr = 58.443 g/mol)
1. Calculate mol, calculate gram (which you would dissolve in
2.   3.7 M Tris-Cl, pH 8 solution (1 Litre) the final buffer for the concentration needed) (Mr given)
3.   EDTA, sodium salt (Mr = 380.2 g/mol) 2. Dilution: C1V1 = C2V2
4.   18 % Sodium dodecyl sulfate solution
3. Calculate mol, calculate gram (which you would dissolve in
5.   Proteinase K solution (177 mg dissolved in 5 ml ddH2O) the final buffer for the concentration needed) (Mr given)
• You need a digestion buffer consisting of the following:
4. Dilution: C1V1 = C2V2
1.   54 mM NaCl
2.   750 uM Tris-Cl, pH 8 5. Dilution: C1V1 = C2V2
3.   42 mM EDTA, pH 8
4.   5.6 % Sodium dodecyl sulfate
5.   160 ug/ml proteinase K
• Calculate: 
• How will you prepare 1260 ml of the digestion buffer? Show all your steps and calculations and remember to explain how
exactly you will make it up.

If molar mass (Mr = __g/mol) is given – most likely calculate mol

 DNA Isolation
• Pre-lecture on efundi
• Demonstration video on efundi
• Students did this at home

• What you need to know about this practical:

• Know why you did each step in that particular sequence
• Know why you used each reagent (what does the reagent do, theory background)
 DNA Spectrophotometry
• Pre-lecture on efundi
• Demonstration video on efundi (DNA purity)
• Students did the practical on-site (DNA purity)

• What you need to know about this practical:

• How the spectrophotometer works (basic principle)
• Know why you used each reagent (what does the reagent do, theory background)
• Absorbance ratio’s
• Why do the compounds absorb light at these wavelengths
• Calculations
 Agarose gel electrophoresis
• Pre-lecture on efundi
• Demonstration video on efundi (How to make a gel)
• Demonstration video on efundi (How to run the gel to check DNA purity)
• Students did the practical on-site
• Linear and coiled DNA cut with restriction enzymes

• What you need to know about this practical:

• How to set up the agarose gel electrophoresis experiment
• Know why you used each reagent (what does the reagent do, theory background)
• Why do we see the DNA fragments at the respective places relative to the ladder?
• Absorbance ratio’s
• Why do the compounds absorb light at these wavelengths
• Calculations
 Tutorial
• Lecture on efundi
Example from the lecture (and study guide, pg 107)

What would the number of and length of the fragments be if

you cut the plasmid with the following restriction enzymes
or combination of enzymes?

NdeI: 1 fragment: 2686 bp

PdmI: 1 fragment: 2686 bp

NdeI & BseYI: 2 fragments: 927 bp + 1759 bp

Given: 183 bp (NdeI); 1110 bp (BseYI); 2686 bp (Total plasmid)
1110 bp -183 bp = 927 bp (fragment 1)
2686 bp – 927 bp = 1759 bp (fragment 2)
 Tutorial
Example from the lecture (and study guide, pg 107)
You want to clone a specific PCR amplicon. You have
determined that the amplicon you want to clone has
enzyme restriction sites for HindIII and EcoRI. After
investigation you have seen that the pUC18/19 plasmid also
have these enzyme restriction sites in its multiple cloning
site (MCS on map above). After enzyme digestion your
amplicon is 854 bp long.

• What length will the recombinant plasmid be after you

have inserted your amplicon? Show your calculation.
Given: 2686 (Total plasmid), 399 bp (HindIII), 455 bp
(EcoRI), 854 bp (amplicon length)
Removed basepairs: 455 bp – 399 bp = 56 bp
2686 – 56 bp + 854 bp = 3484 bp
 Tutorial
Example from the lecture (and study guide, pg 107)
You want to clone a specific PCR amplicon. You have
determined that the amplicon you want to clone has enzyme
restriction sites for HindIII and EcoRI. After investigation you
have seen that the pUC18/19 plasmid also have these
enzyme restriction sites in its multiple cloning site (MCS on
map above). After enzyme digestion your amplicon is 854 bp
long.

• In the amplicon insert you have an enzyme restriction site

for NdeI at 500 bp. If you digest the recombinant plasmid
with this enzyme what length will the fragments be?
Given: 3484 (Total plasmid)
NdeI also cuts your plasmid:
1 enzyme cuts twice  2 fragments
396 (MCS) – 183 (NdeI in plasmid) + 500 (NdeI in amplicon) = 713 bp
3484 – 713 = 2771 bp
Tutorial
You have a circular plasmid (pUC57), which consists of 2710
bp.

• What would the number and length of the fragments be if

you cut the plasmid with the following restriction enzymes
or a combination of enzymes? Show all calculations.
• PdmI & GsuI
• Given: 2318 bp (PdmI); 1808 bp (GsuI); 2710 bp (Total plasmid)
• 2318 bp (PdmI) – 1808 bp (GsuI) = 510 bp (fragment 1)
• 2710 bp – 510 bp = 2200 bp (fragment 2)
Tutorial
You have a circular plasmid (pUC57), which consists of
2710 bp.

You want to clone a specific PCR amplicon. You have

determined that the amplicon you want to clone has
enzyme restriction sites for EcoRI and BamHI. After
investigation you have seen that the pUC57 plasmid also
have these enzyme restriction sites in its multiple cloning
site (MCS on map above, start at 396 bp and end at 476
bp). After enzyme digestion your amplicon is 914 bp long.

Why is 80 bp wrong?

476 bp (MCS end) - 396 bp (MCS start) = 80 bp

Why do you need the whole MCS if your second enzyme
cut MCS in the middle??

If you cut the MCS with EcoRI (start) and HindIII (end) =
maybe 80 bp  You did not use HindIII
 Tutorial
You want to clone a specific PCR amplicon. You have determined that
the amplicon you want to clone has enzyme restriction sites for EcoRI
and BamHI. After investigation you have seen that the pUC57 plasmid
also have these enzyme restriction sites in its multiple cloning site
(MCS on map above, start at 396 bp and end at 476 bp). After
enzyme digestion your amplicon is 914 bp long.

39 bp
Given: 2710 (Total plasmid), EcoRI (no bp given), BamHI
(no bp given), 914 bp (amplicon insert)
Removed basepairs: Physically count = 39 bp

2710 bp – 39 bp + 914 bp = 3585 bp

 Tutorial
You want to clone a specific PCR amplicon. You have determined that
the amplicon you want to clone has enzyme restriction sites for EcoRI
and BamHI. After investigation you have seen that the pUC57 plasmid
also have these enzyme restriction sites in its multiple cloning site
(MCS on map above, start at 396 bp and end at 476 bp). After
enzyme digestion your amplicon is 914 bp long.

In the amplicon insert you have an enzyme restriction site

for NdeI at 864 bp. If you digest the recombinant plasmid
with this enzyme what length will the fragments be?

Given:
3585 bp (Total plasmid),
NdeI cuts plasmid: 183
NdeI cuts amplicon: 864
MCS start at 396 bp
39 bp
2710 bp – 39 bp + 914 bp = 3585 bp
 Tutorial
You want to clone a specific PCR amplicon. You have determined that
the amplicon you want to clone has enzyme restriction sites for EcoRI
and BamHI. After investigation you have seen that the pUC57 plasmid
also have these enzyme restriction sites in its multiple cloning site
(MCS on map above, start at 396 bp and end at 476 bp). After
enzyme digestion your amplicon is 914 bp long.

In the amplicon insert you have an enzyme restriction site

for NdeI at 864 bp. If you digest the recombinant plasmid
with this enzyme what length will the fragments be?

Given:
3585 bp (Total plasmid),
NdeI cuts plasmid: 183
NdeI cuts amplicon: 864
MCS start at 396 bp
39 bp
 Eco RI ? Bp removed
Eco RI
Bam HI
Bam HI
Eco RI Bam HI

914 bp pUC57
2710 bp Remove the digested site
You have an amplicon that you want from plasmid
to clone. It has restriction sites for
Eco RI and Bam HI.

You find a plasmid that have restriction

sites for Eco RI and Bam HI in the MCS Enzyme digestion of the amplicon and
(blue) plasmid with the same enzymes 
Sticky ends

Given (Question b): NdeI

3585 bp (Total plasmid), 1077 NdeI
NdeI cuts plasmid: 183
NdeI cuts amplicon: 864
MCS start at 396 bp
Recombinant pUC57
Fragment 1: Calculate the fragments
? bp
396 – 183 = 213 3585 bp
213 + 864 = 1077

Fragment 2: 2508 Insert the amplicon into

the site where you
3585 – 1077 = 2508 removed bp
Question b: Calculate new plasmid size
Question a: Calculate new plasmid size
If you have any questions regarding
the practical exam, please email me

40834484@nwu.ac.za

or come by at Lab G24.