Team:Toronto/Notebook-w15-tue
console.js:26
Tuesday, August 23
Tuesday, 8/23
Members Present: Alex, Kat, Celine, Karim, Bohdan
Please note, this Benchling entry is longer and more detailed than usual to contain more information than necessary or relative to other entries to describe reason why, purpose of and how protocols were done to update all lab members fully on what is going on and why are plan on doing this.
LAB
●
Miniprep, nanodropped and visualized/gel the previous week ligated products
A | B | C | D | E | |
1 | Sample | 260/280 | 260/230 | ng/uL | Band size (in bp) |
2 | P1A | 1.64 | 7.23 | 10.3 | ~4500 & ~3000 |
3 | P1B | 1.88 | 3.48 | 21.5 | ~4500 & ~3000 |
4 | P1C | 1.87 | 3.43 | 34.4 | ~4500 & ~3000 |
5 | R1A | 1.64 | 0.91 | 139.3 | ~2500 |
6 | R1B | 1.83 | 1.98 | 93.8 | ~2500 |
7 | R1C | 1.82 | 2.44 | 60.8 | ~2500 |
8 | G1A | 1.83 | 2.38 | 20.8 | ~4500 & ~3000 |
9 | G1B | 1.79 | 3.74 | 20.9 | ~4500 & ~3000 |
10 | G1C | 2.18 | -31.18 | 14.1 | ~4500 & ~3000 |
11 | SP1A | 1.77 | 2.24 | 38.5 | ~4000 & ~2500 |
12 | SP1B | 1.86 | 1.84 | 52.6 | ~4000 & ~2500 |
13 | SP1C | 1.82 | 2.04 | 32.2 | ~4000 & ~2500 |
14 | SG1A | 1.77 | 1.67 | 32.6 | ~4000 & ~2500 |
15 | SG1B | 1.8 | 1.85 | 28.6 | ~4000 & ~2500 |
16 | SG1C | 1.85 | 2.21 | 61.8 | ~4000 & ~2500 |
17 | C1A | 1.78 | 1.68 | 68.8 | ~3000 |
18 | C1B | 1.85 | 1.95 | 47.9 | ~3000 |
19 | C1C | 1.85 | 2.67 | 59.9 | ~3000 |
20 | C1 White | 1.89 | 2.03 | 73.4 | ~3000 |
Table1
For understanding notation:
R is pgolB_LacZ. SG is Short_GolS. SP is Short P118A. C is mCherry. P is Long_P118A. G is Long_GolS. The A, B and C beside each sample is a colony from a plate (Colony A, Colony B, Colony C).
○
Miniprep was done using NEB Monarch Miniprep Kit
■
Plasmid Neutralization Buffer was put in the fridge in WB403
■
Anhydrous ethanol was used instead of 95% ethanol in Plasmid Wash Buffer 2
●
Anhydrous ethanol (also known as absolute ethanol) contains relatively low water content (commercial version can contain as low as 0.005% water)
●
The reason why we add ethanol to Plasmid Wash Buffer 2 is to be able to wash and remove residual salts that remain
■
Background information for miniprep:
●
Miniprep is a method isolating plasmid DNA from bacteria
●
Protocol for the NEB Monarch Miniprep Kit can be found here: https://benchling.com/s/prt-tW3NUrMyzrNF2gzbQfqD
○
Nanodrop:
■
Ligation and RE calculator and table:
A | B | C | D | E | F | G | H | I | J | K | L | M | N | O | P | Q | R | S | T | U | V | |
1 | P1A | P1B | P1C | R1A | R1B | R1C | G1A | G1B | G1C | SP1A | SP1B | SP1C | SG1A | SG1B | SG1C | C1A | C1B | C1C | C1 White | |||
2 | Miniprep Concentration | 10.3 | 21.5 | 34.4 | 139.3 | 93.8 | 60.8 | 20.8 | 20.9 | 14.1 | 38.5 | 52.6 | 32.2 | 32.6 | 28.6 | 61.8 | 68.8 | 47.9 | 59.9 | 73.4 | ||
3 | Nuclease-free water | 0 | 0 | 5.37209 | 14.12849964 | 12.73560768 | 10.4211 | 0 | 0 | 0 | 6.61039 | 9.39544 | 4.57764 | 4.73006 | 3.01399 | 10.5275 | 11.186 | 8.64927 | 10.3222 | 11.5504 | ||
4 | DNA | 17 | 17 | 11.6279 | 2.871500359 | 4.264392324 | 6.57895 | 17 | 17 | 17 | 10.3896 | 7.60456 | 12.4224 | 12.2699 | 13.986 | 6.47249 | 5.81395 | 8.35073 | 6.6778 | 5.44959 | ||
5 | Cutsmart | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | ||
6 | Enzyme | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | ||
7 | 20ul | 20ul | 20ul | 20ul | 20ul | 20ul | 20ul | 20ul | 20ul | 20ul | 20ul | 20ul | 20ul | 20ul | 20ul | 20ul | 20ul | 20ul | 20ul | |||
8 | Final DNA Conc. | 8.755 | 18.275 | 20 | 20 | 20.9 | 20 | 17.68 | 17.765 | 11.985 | 20 | 20 | 20 | 20 | 20 | 20 | 20 | 20 | 20 | 20 | ||
Table2
The purpose of this table and the excel chart attached is to calculate how much of the sample/DNA/plasmids you will need to properly do a restriction enzyme digest. In this case for a single restriction enzyme digest, we are aiming for 400ng/uL of DNA in each reaction (so 400/[miniprep] as long as it is below 17, if not you just do 17 as your final volume must be 20ul)
■
Pictures of nanodrop graphs are after the gel
○
Gel:
■
Used 9.8ul of sample and 6ul for DNA (NEB 2-log) ladder at 100V, 3.00A, 300W for 1 hour
Gel 1:
Lane 1: NEB 2-Log DNA Ladder
Lane 2: R1A, 1 band at ~2500bp. Expected band size for pgolB_LacZ is ~2419bp. (This is a good band)
Lane 3: R1B, 1 band at ~2500bp. Expected band size for pgolB_LacZ is ~2419bp. (This is a good band)
Lane 4: R1C, 1 band at ~2500bp. Expected band size for pgolB_LacZ is ~2419bp. (This is a good band)
Lane 5: G1A, 2 bands at ~4500bp and ~3000bp. Expected band size for Long_GolS is ~2999bp (This is good bands)
Lane 6: G1B, 2 bands at ~4500bp and ~3000bp. Expected band size for Long_GolS is ~2999bp (This is good bands)
Lane 7: G1C, 2 bands at ~4500bp and ~3000bp. Expected band size for Long_GolS is ~2999bp (This is good bands)
Lane 8: P1A, 2 bands at ~4500bp and ~3000bp. Expected band size for Long_P118A is ~3070bp (This is good bands)
Lane 9: P1B, 2 bands at ~4500bp and ~3000bp. Expected band size for Long_P118A is ~3070bp (This is good bands)
Lane 10: P1C, 2 bands at ~4500bp and ~3000bp. Expected band size for Long_P118A is ~3070bp (This is good bands)
Lane 11: SP1A, 2 bands at ~4000bp and ~2500bp. Expected band size for Short_P118A is ~2670bp (This is good bands)
Lane 12: SP1B, 2 bands at ~4000bp and ~2500bp. Expected band size for Short_P118A is ~2670bp (This is good bands)
Lane 13: SP1C, 2 bands at ~4000bp and ~2500bp. Expected band size for Short_P118A is ~2670bp (This is good bands)
Lane 14: NEB 2-Log DNA Ladder
Gel 2:
Lane 1: NEB 2-Log DNA Ladder
Lane 2: SG1A, 2 bands at ~4000bp and ~2500bp. Expected band size for Short_GolS is ~2662bp (this is good bands)
Lane 3: SG1B, 2 bands at ~4000bp and ~2500bp. Expected band size for Short_GolS is ~2662bp (this is good bands)
Lane 4: SG1C, 2 bands at ~4000bp and ~2500bp. Expected band size for Short_GolS is ~2662bp (this is good bands)
Lane 5: C1A, 1 band at ~3000bp. Expected band for mCherry is ~2916bp (This is a good band)
Lane 6: C1B, 1 band at ~3000bp. Expected band for mCherry is ~2916bp (This is a good band)
Lane 7: C1C, 1 band at ~3000bp. Expected band for mCherry is ~2916bp (This is a good band)
Lane 8: C1White, 1 band at ~3000bp. Expected band for mCherry is ~2916bp (though do remember, the white in the name indicates the colony was white but was suppose to be red due to the mCherry but due to it having a different appearance but yet same band size, this could indicate that a mutation occurred within this colony)
Lane 9: NEB 2-Log DNA Ladder
○
To find estimated band length, you just add 2070bp (the approximate size of the backbone) to the size of each gBlock linear plasmid
■
The size of each plasmid can be determined by looking at the gBlock plasmids we created (I'll link below) minus 61bp which are UNS sites (Unique Nucleotide Sequences)
●
R1/pgolB_LacZ plasmid: https://benchling.com/s/dWfmQsPX
●
G1/Long_GolS plasmid: https://benchling.com/s/xpyFCul4
●
P1/Long_P118A plasmid: https://benchling.com/s/nPYbrk4R
●
SP1/Short_P118A plasmid: https://benchling.com/s/3m7f2WXW
●
SG1/Shrot_GolS plasmid: https://benchling.com/s/Y34ZfUg5
●
C1/mCherry plasmid: https://benchling.com/s/PB0U08XQ
○
Gel troubleshooting:
■
A lot can happen during a gel so before the gel, best try and work on preventing things like making sure proper TAE concentration was made (and that it wasn't too hot when poured), right voltage, watts, amps and duration, gel going right direction, etc...
■
For the actual bands, many different things can happen and many different explanations:
●
Smeared bands like we had in the past:
a.
They are undigested DNA/samples
b.
DNA began to degrade
c.
Too much DNA/sample was added
d.
High salt concentration
e.
Bad wells
●
Guide to troubleshooting gels:
○
https://drive.google.com/file/d/0B6Z4m6vl-WRuSXB0N25rTmwxS00/view?usp=sharing
●
Nanodrop graph results:
○
C1A and C1B are not included because we forgot to save pictures of it
○
For the reference of understanding and interpretation:
■
260/280 is the absorbance at 260 and 280 nm
●
Used to assess the purity of RNA and RNA
●
Ratio of ~1.8 is generally accepted as "pure" for DNA
●
Ratio of ~2.0 is generally accepted as "pure" for RNA
●
Ratio is appreciably lower than ~1.8, then it indicates presence of protein, phenol or other contamination
■
260/230 is the absorbance at 260 and 230nm
●
Secondary measure of nucleic acid purity
●
For "pure" nucleic acid, often higher than respective 260/280 values
○
Commonly in range of 1.8-2.2
○
If ratio is appreciably lower, may indicate presence of co-purified contaminants
■
ng/uL
●
Sample concentration in ng/ul based on absorbance at 260nm and selected analysis constants
G1C:
ng/uL = 14.1
260/280 = 2.18 (Indicates closer absorbance to RNA rather than DNA but due to G1 A and B both indicating DNA, it is safe to assume that G1 is still good. For future reference to troubleshooting; acidity can cause decreased 260/280 ratios and basic-ness can cause increase of 260/280 ratios, spectro not always perfect so if you ever have time or enough sample you should try redoing the same sample)
260/230 = -31.18 (Bad result. Negative ratios can be caused by the blank having a higher absorbance than your sample)
P1A:
ng/uL = 10.3
260/280 = 1.64 (Usually you assume content based if it is ~0.2 to or from the estimated value, ~1.8 or ~2.0. Due to it being within that deviation, we would assume it is "pure" DNA)
260/230 = 7.23 (When a 260/230 is high, it is usually recommended to retest the nanodrop and see if it changes. If it does not change, it can indicate that the sample is too diluted or there is an rather low concentration of phenols and salt.)
Administrative:
●
All wet lab members were instructed to register for the iGEM team roster (if you have not yet and do not know how, please contact Alex, Seray or Anthony)
TO DO:
For the next day:
LAB TEAM:
●
Gel extract the samples with two bands
●
Create primers (anyone wanna help do it with Alex?)
●
If cells are not competent, we are going to make more
LAB MANAGERS:
●
Dish washing (sorry)
●
Pipette tip boxes need refill and autoclaving (sorry again)
●
Need to buy more NEB 2-Log DNA Ladder
References:
●
Nanodrop User Manual: http://www.nanodrop.com/library/nd-1000-v3.7-users-manual-8.5x11.pdf
●
Readings into UNS: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4899833/
