MODX LAB PRACS Flashcards
(55 cards)
1
Q
Master mix composition (reverse transcriptase qPCR):
A
- Taq polymerase (DNA polymerase)
- Probe
- Free nucleotides
- Forward primer
- Reverse primer
- Reverse Transcriptase
2
Q
In BGI Test Kit, The”master mix” consists of the reagents:
A
- reaction mix and enzyme mix
3
Q
reaction mix is composed of:
A
- Probe
- Primers
- Free nucleotides
4
Q
enzyme mix is composed of:
A
- Taq polymerase
- Reverse Transcriptase
5
Q
Negative Control is composed of
A
Master Mix + Blank
6
Q
Reagent Blank is made of?
A
Master Mix
7
Q
The Reagent Blank is also known as?
A
NTC (No Template Control)
8
Q
Vial with red cap is for?
A
Enzyme Mix
9
Q
Vial with green cap is for?
A
Reaction mix
10
Q
Vial with white cap is for?
A
Positive control
11
Q
Vial with black cap is for?
A
Water or Blank
12
Q
What should you put in the first well?
A
Reagent Blank
13
Q
Why do we need to have 2 Negative control?
A
To increase the sensitivity against contamination
14
Q
Is a sequence of DNA, RNA, or protein that represents the most common nucleotide or amino acid at each position form a set of aligned sequences.
A
CONSENSUS SEQUENCE
15
Q
purpose of consensus sequences
A
- identify the common or important feature shared among similar DNA, RNA, or protein sequences.
- To increase the accuracy and reduce the errors during DNA sequencing
16
Q
Recommended for nucleotide alignments
A
MAFFT
17
Q
faster, less precise, can reverse sequences
A
MAFFT
18
Q
Recommended for amino acid alignments
A
Clustal Omega
19
Q
Slower, more precise, cannot reverse sequences
A
Clustal omega
20
Q
standard lab procedure for separating DNA by size for visualization and purification.
A
Gel electrophoresis
21
Q
Principle of Gel electrophoresis
A
uses an electrical field to move the negatively-charged DNA through an agarose gel matrix toward a positive electrode.
22
Q
________ DNA fragments migrate through the gel more quickly than _____ ones.
A
Shorter. Longer
23
Q
HOW TO CREATE TAE BUFFER (50X):
A
- Add 242 g of Tris base
- Add 57.1 mL of Acetate
(100% acetic acid) - Add 100 mL of 0.5M sodium EDTA
24
Q
purifying the DNA for later use:
A
use long- wavelength UV and expose for as little time as possible
25
HOW TO GET BETTER RESOLUTION (CRISPNESS) OF THE BANDS?
1.Running the gel at a lower voltage for a longer period of time
2.Using a wider/thinner gel comb
3.Loading less DNA into the well.
4.Choose the optimal agarose concentration.
5.Choose the right buffer. TAE vs. TBE.
6.Choose a proper sample loading dye/buffer.
7.Choose the optimal sample quantity. (EtBr=20ng/well;SyBr=1 ng/well)
8.Choose the optimal gel size. (For small gels: 8 x 10 cm gels. 30–50 mL. For larger
gels: Southern and northern blotting.250 mL.)
9.Avoid the “smiling” effect
10.Gel must be fully submerged
26
optimal sample quantity: EtBr ; SyBr
EtBr 20ng/ well ; SyBr 1ng/ well
27
optimal gel size
For small gels: 8 x 10 cm gels.
30–50 mL. For larger gels
Southern and northern blotting.250 mL.
28
Too much DNA loaded on a gel can affect
the migration of the sample
29
Too little DNA is?
hard to
detect on a gel
30
an overloaded fragment
runs slower hence, seem to be larger in size
than it really is.
31
What is the remedy of Uneven heating of the gel across different lanes caused by high voltage.
Remedy: reduce the voltage.
32
Uneven distribution of the electric field across the gel width.
Remedy: checking the tank setup for loose contacts
33
sample remains in the gel well
due to overloading and protein contam
34
sample floats after loading
incorrect loading buffer
35
speckles in the gel
flueorescing contaminants
36
Low quantity of sample, it can bedifficult to visualize the bands. What are the remedies?
- recommended to load a minimum of 0.1-0.2 ug of DNA or RNA sample per millimeter of gel well width.
- Use a gel comb with deep and narrow wells
37
Remedy in Sample degradation
- Make sure reagents selected are molecular biology grade
- labware is free of nucleases.
- Proper PPE
38
Remedy in Loading dye masking the desired band
Check the apparent migration size of the loading dye(s) used in electrophoresis.
The dye may mask and hide the bands of interest, especially when the sample volume is low
39
Remedy in Bubbles introduced during sample loading
make sure air bubbles are not trapped in the well during sample loading.
40
Remedy in Well damaged during sample loading
Avoid puncturing the wells with the pipette tips during sample loading.
41
Remedy in Sample wells containing residual acrylamide and/or urea
When using polyacrylamide gels, flush residual acrylamide (and urea, in the case of denaturing gels) out of the wells, prior to sample loading.
42
Remedy in Very low or high voltage
Apply voltage as recommended for the size range of the nucleic acids and the running buffer used.
43
Remedy in Very short or long run time
Run the gel long enough to ensure bands are resolved sufficiently. However, a very long run may generate excessive heat, denature samples, and cause bands to diffuse.
44
Sample overloading
Do not overload samples in wells; the general recommendation is 0.1-0.2 pg of sample per millimeter of a gel well's width. Trailing smears, warped or U-shaped bands, and bands that appear fused are a common characteristic of overloaded gels.
45
Sample in high-salt buffer
- Check that the loading buffer's salt concentration is compatible with the selected gel. Dilute the loading buffer, if necessary.
- If the nucleic acid sample is already in a high-salt buffer, dilute the sample in nuclease-free water before adding the loading buffer. If needed, purify or precipitate the nucleic acid sample and resuspend it in nuclease-free water to remove excess salt.
46
Sample containing high amounts of protein
- Proteins present in the sample may interfere with sample mobility in the gel. Remove the protein by purifying the sample, or dissociate/denature the protein by preparing the sample in a loading dye with SDS and heating it before loading.
47
Incompatible loading buffer
• For electrophoresis of single-stranded nucleic acids, use a loading dye containing a denaturant and then heat the sample, to prevent formation of undesirable duplexes.
• For electrophoresis of double-stranded DNA, avoid a loading dye with denaturant and do not heat the sample, to preserve the duplex structure.
48
Incorrect gel percentage
adjust the gel volume with water after boiling to compensate for evaporation; this prevents the increase in gel percentage than intended.
49
Suboptimal gel choice
Choose a gel type that works better to separate the samples. Polyacrylamide gels are recommended for resolving nucleic acids <1,000 bp.
50
Poorly formed wells
- Clean the gel comb properly before using it in casting the gel.
- do not push the comb all the way to the bottom of the gel.Avoid overfilling the gel tray,- wells.
- Allow sufficient time for the wells to form before removing the comb. Once the gel is solidified, remove the comb carefully and steadily to prevent damage to the wells.
51
Incorrect gel type
For electrophoresis of single-stranded nucleic acids such as RNA, prepare a denaturing gel for efficient separation.
For electrophoresis of double-stranded DNA, avoid using denaturing gels to preserve the duplex structure.
52
Incompatible running buffer
Make sure the buffer used works well with the samples to be separated.
53
Very high voltage
Avoid using more than the recommended voltage, as it could lead to excessive heat generation,
54
Excessive heat generation
- Ensure that the running buffer has high buffering capacity,
- cool the gel apparatus when appropriate. Lower the voltage,
55
Let agarose solution cool down to about? _____
50C, takes about 5 mins
