11: PCR and Electrophoresis

Students extract DNA, perform initial and nested PCR amplification of the GAPDH gene, run gel electrophoresis to separate DNA fragments, and interpret band patterns.

Gene Expression Fold Change from RT-qPCR Data

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	Untreated Cells		Cells Treated with Experimental Drug
	House Keeping Gene	Gene of Interest	House Keeping Gene	Gene of Interest
Well 1	19.5	21.4	19.4	21.2
Well 2	19.5	22.6	19.1	21.1
Well 3	18.9	22.9	18.6	21.1
mean Ct	19.3	22.3	19.0	21.1
ΔCt
ΔΔCt

Given the data in the table above calculate the fold change (2^|ΔΔCt|) value for the effect of the drug on the cells.
Note: answers need to be within 3% of the correct number to be correct.

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DNA Fragment Migration in Agarose Gel Electrophoresis (Closest/Farthest)

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Which one of the following fragments of DNA would migrate closest to the wells during agarose gel electrophoresis?

• A. 95 base pairs
• B. 400 base pairs
• C. 3,000 base pairs
• D. 6,500 base pairs

Check Answer

 

DNA Fragment Size from Agarose Gel Migration (Numeric)

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Gel Migration Problem

DNA Marker	# of Base Pairs (bp)	Migration Distance (cm)
500 base pairs	500	2.84
1,000 base pairs	1000	2.42
2,000 base pairs	2000	2.01
3,000 base pairs	3000	1.77
5,000 base pairs	5000	1.46
10,000 base pairs	10000	1.04
Unknown	? ?	1.29

The standard DNA ladder and unknown DNA strand listed in the table were separated using an agarose gel
Estimate the number of base pairs of the unknown DNA strand.
Note: answers need to be within 19% of the correct number to be correct.

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Inverse PCR Primer Selection (15 nt)

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EcoR1 overhang	left unknown sequence	central known sequence	right unknown sequence	EcoR1 overhang
5′–      A   A   ,   T   T   C  – 3′–              ,           G  –	–  N   N   N   ,   N   N   N   ,   N   N   N  – –  N   N   N   ,   N   N   N   ,   N   N   N  –	–  A   G   T   ,   G   A   C   ,   C   T   C   ,   A   G   G   ,   T   C   C  – –  T   C   A   ,   C   T   G   ,   G   A   G   ,   T   C   C   ,   A   G   G  –	–  N   N   N   ,   N   N   N   ,   N   N   N  – –  N   N   N   ,   N   N   N   ,   N   N   N  –	–  G           ,              –3′ –  C   T   T   ,   A   A      –5′

Choose the correct pair of RNA primers that will amplify the both the known and unknown region of DNA shown above using inverse PCR.
The RNA primers are 6 bases in length.
Pay close attention to the 5′ and 3′ ends of the primers.

• A.  

  5′–

  U

  C

  A

  ,

  C

  U

  G

  –3′

  AND  

  5′–

  C

  C

  U

  ,

  G

  G

  A

  –3′

• B.  

  5′–

  A

  G

  G

  ,

  U

  C

  C

  –3′

  AND  

  5′–

  U

  C

  A

  ,

  C

  U

  G

  –3′

• C.  

  5′–

  G

  U

  C

  ,

  A

  C

  U

  –3′

  AND  

  5′–

  C

  C

  U

  ,

  G

  G

  A

  –3′

• D.  

  5′–

  C

  A

  G

  ,

  U

  G

  A

  –3′

  AND  

  5′–

  A

  G

  G

  ,

  U

  C

  C

  –3′

• E.  

  5′–

  A

  G

  G

  ,

  U

  C

  C

  –3′

  AND  

  5′–

  U

  C

  C

  ,

  A

  G

  G

  –3′

• F.  

  5′–

  G

  U

  C

  ,

  A

  C

  U

  –3′

  AND  

  5′–

  A

  G

  G

  ,

  U

  C

  C

  –3′

Check Answer

 

Protein Molecular Weights from SDS-PAGE Band Positions

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Lane 1 shows a simulated Kaleidoscope-style pre-stained protein ladder.

Match each colored band to its molecular weight.

	Your Choice	Prompt
	Drop Your Choice Here	1.  colored band
	Drop Your Choice Here	2.  colored band
	Drop Your Choice Here	3.  colored band
	Drop Your Choice Here	4.  colored band
	Drop Your Choice Here	5.  colored band

Drag one of the choices below:

• A. 250 kDa
• B. 37 kDa
• C. 10 kDa
• D. 150 kDa
• E. 25 kDa

Check Answer Reset Game

 

Protein Molecular Weight from an SDS-PAGE Ladder

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Below is a simulated SDS–PAGE gel.
Lane 1 contains a Kaleidoscope-style pre-stained protein ladder. Lane 2 contains a single protein band.
The gel was run for too short a time (bands are compressed near the top).
Standard ladder reference (kDa):

			– 250
			– 150
			– 100
			– 75
			– 50
			– 37
			– 25
			– 20
			– 15
			– 10

Gel results:

Lane 1

Lane 2

What is the molecular weight (kDa) of the band in lane 2?
Assume ln(MW) is approximately linear with migration distance.
Note: answers need to be within 10% of the correct number to be correct.

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Restriction Fragment Sizes (12 Length, 2 Sites)

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DNA Fragment Question: Shown below is a short DNA fragment that is only 12 kb in length. This fragment has been isolated for restriction enzyme analysis.

					SspI								BlnI						SspI
	0		1		2		3		4		5		6		7		8		9		10		11		12

Two (2) distinct types of restriction enzyme recognition sites, SspI and BlnI, are labeled at the top of this DNA segment.

Determine the sizes of the DNA bands that would appear on an agarose gel after digestion with SspI only.

• A. 1 kb
• B. 2 kb
• C. 3 kb
• D. 4 kb
• E. 5 kb
• F. 6 kb
• G. 7 kb
• H. 8 kb

Check Answer Clear Selection

 

Nested PCR Primer Pair Selection (24 nt)

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5′–  G   C   T   ,   T   G   A  – 3′–  C   G   A   ,   A   C   T  –

–  G   A   C   ,   T   G   G   ,   A   A   A   ,   G   A   G   ,   T   A   A   ,   C   G   T   ,   A   G   T   ,   C   A   A  – –  C   T   G   ,   A   C   C   ,   T   T   T   ,   C   T   C   ,   A   T   T   ,   G   C   A   ,   T   C   A   ,   G   T   T  –

–  A   C   G   ,   T   A   C  –3′ –  T   G   C   ,   A   T   G  –5′

The amplicon sequence of DNA shown above was replicated using 30 cycles of PCR, using the primers 5′-GCTTGA-3′ and 5′-GTACGT-3′.
But the first PCR run contained significant contamination due to mispriming. Probably from using too short of primers that were only 6 nucleotide in length.
Choose the correct pair of RNA primers that will amplify the remaining region of DNA inside the old primers using nested PCR. The nested RNA primers are 6 bases in length.
Pay close attention to the 5′ and 3′ ends of the primers.

• A.  

  5′–

  U

  U

  G

  ,

  A

  C

  U

  –3′

  AND  

  5′–

  G

  G

  U

  ,

  C

  A

  G

  –3′

• B.  

  5′–

  G

  G

  U

  ,

  C

  A

  G

  –3′

  AND  

  5′–

  U

  C

  A

  ,

  G

  U

  U

  –3′

• C.  

  5′–

  U

  U

  G

  ,

  A

  C

  U

  –3′

  AND  

  5′–

  C

  C

  A

  ,

  G

  U

  C

  –3′

• D.  

  5′–

  G

  A

  C

  ,

  U

  G

  G

  –3′

  AND  

  5′–

  U

  C

  A

  ,

  G

  U

  U

  –3′

• E.  

  5′–

  U

  C

  A

  ,

  G

  U

  U

  –3′

  AND  

  5′–

  A

  G

  U

  ,

  C

  A

  A

  –3′

• F.  

  5′–

  G

  A

  C

  ,

  U

  G

  G

  –3′

  AND  

  5′–

  U

  U

  G

  ,

  A

  C

  U

  –3′

Check Answer

 

PCR Primer Selection (36 bp Template, 9-nt Primers)

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5′–

A

C

G

,

A

T

C

,

G

A

A

,

C

T

T

,

A

G

G

,

C

T

C

,

T

G

G

,

A

G

T

,

A

G

G

,

A

C

A

,

T

C

G

,

A

C

C

–3′

3′–

T

G

C

,

T

A

G

,

C

T

T

,

G

A

A

,

T

C

C

,

G

A

G

,

A

C

C

,

T

C

A

,

T

C

C

,

T

G

T

,

A

G

C

,

T

G

G

–5′

Choose the correct pair of RNA primers that will amplify the entire region of DNA shown above using PCR. The RNA primers are 9 bases in length.
Pay close attention to the 5′ and 3′ ends of the primers.

• A.  

  5′–

  G

  G

  U

  ,

  C

  G

  A

  ,

  U

  G

  U

  –3′

  AND  

  5′–

  A

  C

  A

  ,

  U

  C

  G

  ,

  A

  C

  C

  –3′

• B.  

  5′–

  U

  G

  C

  ,

  U

  A

  G

  ,

  C

  U

  U

  –3′

  AND  

  5′–

  U

  U

  C

  ,

  G

  A

  U

  ,

  C

  G

  U

  –3′

• C.  

  5′–

  G

  G

  U

  ,

  C

  G

  A

  ,

  U

  G

  U

  –3′

  AND  

  5′–

  A

  A

  G

  ,

  C

  U

  A

  ,

  G

  C

  A

  –3′

• D.  

  5′–

  A

  C

  G

  ,

  A

  U

  C

  ,

  G

  A

  A

  –3′

  AND  

  5′–

  U

  G

  U

  ,

  A

  G

  C

  ,

  U

  G

  G

  –3′

• E.  

  5′–

  A

  C

  G

  ,

  A

  U

  C

  ,

  G

  A

  A

  –3′

  AND  

  5′–

  G

  G

  U

  ,

  C

  G

  A

  ,

  U

  G

  U

  –3′

• F.  

  5′–

  A

  A

  G

  ,

  C

  U

  A

  ,

  G

  C

  A

  –3′

  AND  

  5′–

  U

  G

  U

  ,

  A

  G

  C

  ,

  U

  G

  G

  –3′

Check Answer

 

Protein Molecular Weight from SDS-PAGE Migration

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Gel Migration Problem

In this task, data from an SDS-PAGE experiment, where proteins are separated based on molecular weight, is provided. The gel results table below shows some standard proteins with known molecular weights and one unknown protein.

Protein Name	Molecular Weight (kDa)	Migration Distance (cm)
Hemoglobin (Hem)	16.7	3.25
Ferritin (Fer)	19.8	3.11
Chymotrypsin (Chy)	25.0	2.92
Lipase (Lip)	40.0	2.55
Enolase (Eno)	42.5	2.50
Sucrase (Suc)	51.0	2.35
Fibrinogen (Fib)	63.5	2.18
Unknown	?	2.24

Estimate the molecular weight of the unknown protein by comparing its gel migration distance with those of the standards.
Note: answers need to be within 6% of the correct number to be correct.

Check Answer