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===============================================================================
Fenner Macrae Megadaph - Mitochondrial Analysis Lab Notebook
===============================================================================
This is continued from a non-digital lab notebook 12/3/16 - 8/22/17
===============================================================================
August 22nd, 2017
===============================================================================
-------------------------------------------------------------------------------
Meeting with Sarah
-------------------------------------------------------------------------------
Discussed primer design for amplifying the control region in order to resolve
the conflicting results which we're finding for the NOVOplasty assemblies. Plan
is to amplify in all 9 D. magna starting controls. We should be able to tell by
the fragment size whether the large repetitive mtDNA assemblies are correct.
-------------------------------------------------------------------------------
Primer Design
-------------------------------------------------------------------------------
-Used the NOVOPlasty assembly for IB as the reference
(fmacrae/daphnia_mitochondria_MA/illumina_magna_150bp/I/processed/\
create_consensus/IB)
- First trimmed the IB assembly down to only the ranges 11812:12738 and
555:1541 which should correspond roughly to the ND1 and ND2 genes
respectively.
- Then moved ND1 region in front of the ND2 region and placed a run of Ns in
between.
- Then used web interface of primer3 to choose primers (Max product size 400bp)
(local: proj/research/daphnia_mtdna_ma/megadaph.mtdna/data-raw/primer_design/\
primer3_out.html).
- Using jalview I Muscle aligned the consensus sequences of all isolates with
the ND1 and ND2 sequence.
- Found primers which look good:
(local: proj/research/daphnia_mtdna_ma/megadaph.mtdna/data-raw/primer_design/\
(primers.control_region.fasta)
- Sarah Ok'd the primers.
- While looking into known info about D. magna mtDNA structure we found a very
interesting looking paper about D. galeata mtDNA diversity in Japan (2017). We
had to contact the authors directly to access the paper.
- Also contacted Dieter about whether he has PacBio data for the D. magna
mtDNA.
===============================================================================
August 23rd, 2017
===============================================================================
- Created an R package fen.R.util (https://github.com/fennerm/fen.R.util) to
house code shared between other R packages.
===============================================================================
August 28th, 2017
===============================================================================
- Large reorganization of project github. Local and IBEST directories should
now be kept fairly well in sync using github. Data will still reside only on
IBEST.
===============================================================================
August 30th, 2017
===============================================================================
- Setup my personal python modules as a package (fmbiopy) for easier import and
extensibility.
- Using pytest for package testing (https://docs.pytest.org/en/latest/)
===============================================================================
September 5th, 2017
===============================================================================
- Received a reference sequence for one of the European D. magna clones from
Dieter Ebert's lab. It is 14969 bp, just 20 more than the Chinese reference.
Edited the novoplasty_assemble.py script to use this reference, and bounded it
between 14000-16000 bp. Started running it on the F isolates as a test run.
- Still getting too large/too small assemblies. Edited novoplasty_assemble.py
to also use the given seed as a reference sequence.
===============================================================================
September 6th, 2017
===============================================================================
- Using the fasta sequence as a reference in NOVOplasty seems to make no
difference. Next I'll try just aligning directly to this reference and see if
that works well enough
- Aligned downsampled FA10 reads to the European mtDNA sequence.
- Rotated alignments still looking weird. Large drop in coverage at the split
point then a large increase in coverage. All the reads which align across the
split point are F2F1 oriented.
- Talked to Sarah. We decided to go ahead with Sanger sequencing of the control
regions, and ask Anna for advice.
===============================================================================
September 11th, 2017
===============================================================================
- Sarah checked my primers and decided to used alternatives due to bad GC
content in the ones I suggested. Two sets were ordered. Need to check that we
have everything else for the reaction in lab.
===============================================================================
September 13th, 2017
===============================================================================
- Sarah said she'd ask about long-range Taq primers for sequencing of control
region. Derek volunteered some.
- Lab meeting: Introductions - my presentation slot set for Nov. 29th
===============================================================================
September 14th-15th, 2017
===============================================================================
------------------------------------------------------------------------------
Extraction and PCR 2
------------------------------------------------------------------------------
-------------------------------------------------------------------------------
DNA extraction protocol:
-------------------------------------------------------------------------------
- Isolate 5 German and 5 Israeli D. magna (IA10-6 and GB4-19)
- Pipet off extra water
- Crush up Daphnia.
- Extract DNA using Zymo Quick-DNA Universal Kit - protocol saved locally to
~/proj/research/daphnia_mtdna_ma/docs/protocols/\
Zymo_Daphnia_DNA_Extraction_Protocol.pdf
-------------------------------------------------------------------------------
Nanodrop Readings:
-------------------------------------------------------------------------------
GB4-19
------
Conc.: 11.2 ng/uL (817.6 ng in 73 uL)
Abs: 0.351
A-260: 0.225
A-280: 0.148
260/280: 1.52
260/230: 0.64
IA10-6
------
Conc.: 9.8 ng/uL (715.4 ng in 73 uL)
Abs: 0.252
A-260: 0.225
A-280: 0.148
260/280: 1.52
260/230: 0.64
-------------------------------------------------------------------------------
Amplification protocol:
-------------------------------------------------------------------------------
Samples:
-(2 isolates + 1 no template negative controls)
x 2 primers
Primers:
- Primers 1 = fwd: TAGGGTTGCCCTTGTTTTTG, rev: GGACCCTACCTCGATTTCGT
- Primers 2 = fwd: TAGGGTTGCCCTTGTTTTTG, rev: GGTACGTGGGACCCTACCTC
- Use KOD hot start DNA polymerase and follow associated protocol. Saved
locally to:
~/proj/research/daphnia_mtdna_ma/docs/protocols/kod_hot_start_polymerase.pdf
- Possible PCR product sizes: 4500bp - 7200bp
- Possible things to vary if reaction goes wrong:
- ng of template gDNA
- Amount of MgSO4
- Different annealing temps
- Extension times - Try 140s
- # of Cycles
Reference: KOD Hot Start Polymerase protocol
PCR 1
-----
(2 samples + 1 negative control) x 2 primers
Sample IDs:
1 - G, primer 1
2 - G, primer 2
3 - I, primer 1
4 - I, primer 2
5 - control, primer 1
6 - control, primer 2
Mix 1
-----
- 2.5uL 10X Standard Taq buffer
- 1.5uL 25mM MgSO4
- 2.5uL dNTPs (2 mM each)
- 15.5uL Nanopure water
- 0.75uL Fwd Primer (10 uM)
- 0.75uL Rev Primer (10 uM)
- 1uL Template (~20ng gDNA)
- 0.5uL KOD Hot Start Polymerase
Total: 25uL
Program 1
---------
Activation 95C for 2 min
(
Denature 95C for 20s
Annealing 53.5C for 10s
Extension 70C for 180s
) x 30 cycles
-------------------------------------------------------------------------------
Gel protocol:
-------------------------------------------------------------------------------
- Run at < 70V
- 20 uL reactions - use 5 or 6uL for gel.
Gel recipe
----------
1 g agarose in 100 mL 1X TBE
Loading Samples
---------------
Ladder:
3uL Elution Buffer, 1uL EZ vision 3 dye, 2uL NEB 1kb ladder
Samples:
2uL EZ vision 3 dye, 10uL PCR product
Layout
------
From right to left (with grooved-side of gel facing you)
Lane Sample ID
~~~~~~~~~~~~~~~~~
R
.
.
| 6
| 5
| 4
| 3
| 2
| 1
| Ladder
L
Gel Results
-----------
Failed. Smearing of large fragments in all samples including negative controls.
I think most likely failure point is extraction due to the low nanodrop
readings. Will redo extraction on fresh animals, and try to be more careful
about sterile technique. Also possible that extension times are too long. Will
try and vary next time.
===============================================================================
September 17th-18th, 2017
===============================================================================
DNA extractions carried out on 5 Daphnia from IB3 and GB8.
------------------------------------------------------------------------------
Extraction and PCR 2
------------------------------------------------------------------------------
-------------------------------------------------------------------------------
Nanodrop Readings:
-------------------------------------------------------------------------------
GB8
------
Conc.: 19.8
Abs: 0.614
A-260: 0.397
A-280: 0.259
260/280: 1.53
260/230: 0.65
IB10
------
Conc.: 53.5
Abs: 1.747
A-260: 1.069
A-280: 0.722
260/280: 1.48
260/230: 0.61
-------------------------------------------------------------------------------
Amplification protocol:
-------------------------------------------------------------------------------
PCR 2
-----
(2 samples + 1 extraction neg control) x 2 primers
Sample IDs:
1 - G, primer 1
2 - G, primer 2
3 - I, primer 1
4 - I, primer 2
5 - control, primer 1
6 - control, primer 2
Mix 2
-----
(Uses MgCl2 rather than MgSO4)
- 2.5uL 10X Standard Taq buffer
- 1.5uL 25mM MgCl2
- 2.5uL dNTPs (2 mM each)
- 15.5uL Nuclease Free water
- 0.75uL Fwd Primer (10 uM)
- 0.75uL Rev Primer (10 uM)
- 1uL Template (~20ng gDNA)
- 0.5uL KOD Hot Start Polymerase
Total: 25uL
Same program as PCR 1 (Program 1)
-------------------------------------------------------------------------------
Gel protocol:
-------------------------------------------------------------------------------
- Run at < 70V
- 20 uL reactions - use 5 or 6uL for gel.
Gel recipe
----------
1 g agarose in 100 mL 1X TBE run at 70V
Loading Samples
---------------
Ladder:
3uL Elution Buffer, 1uL EZ vision 3 dye, 2uL NEB 1kb ladder
Samples:
1uL EZ vision 3 dye, 5uL PCR product
Layout
------
From left to right (with grooved-side of gel facing you)
Lane Sample ID
~~~~~~~~~~~~~~~~~
L
| .
| .
| .
| .
| 1
| 2
| 3
| 4
| 5
| 6
| Ladder
| .
| .
| .
| .
R
Gel Results
-----------
Another failure - more promising, but dumber reason.
- Used the primer stocks rather than the diluted aliquots so the primer the
gel just shows super bright bands at low fragment size. 5 looks like the
primers are missing (another sloppy mistake). In 3 there is a smear across
almost the entire lane, 1 also has a smear but less bright.
-------------------------------------------------------------------------------
PCR 3
-------------------------------------------------------------------------------
PCR reaction recipe same as above but with correct primer concentrations
(Mix 2, Program 1)
Gel recipe
----------
1 g agarose in 100 mL 1X TBE run at 70V
Loading Samples
---------------
Ladder:
3uL Elution Buffer, 1uL EZ vision 3 dye, 2uL NEB 1kb ladder
Samples:
1uL EZ vision 3 dye, 5uL PCR product
Layout
------
From left to right (with grooved-side of gel facing you)
Lane Sample ID
~~~~~~~~~~~~~~~~~
L
| .
| .
| .
| .
| Ladder
| 1
| 2
| 3
| 4
| 5
| 6
| .
| .
| .
| .
R
Results
-------
No bands except ladder. Sarah ordered positive control primers - we'll wait for
those to arrive before doing more troubleshooting.
===============================================================================
September 21st, 2017
===============================================================================
- Received positive control primers which amplify 16s rRNA. Diluted to 100uM
then made 10uM PCR stocks (in Tris)
- PC Primers = fwd: AGAAAGCCACCCCTACTAAAA, rev: GGTTAACCCTTTGGAGCTTT
- Nanodrop DNA concentration for positive control DNA: 12.4 ng/uL
-------------------------------------------------------------------------------
PCR 4
-------------------------------------------------------------------------------
This time vary extension time and run positive controls (PC).
Used different amounts of template to include 50ng gDNA as recommended by the
KOD protocol
Mix 3
-----
- 2.5uL 10X Standard Taq buffer
- 1.5uL 25mM MgCl2
- 2.5uL dNTPs (2 mM each)
- Nuclease Free Water: 12.5uL (PC)/ 14 uL (G) / 15.5uL (I)
- 0.75uL Fwd Primer (10 uM)
- 0.75uL Rev Primer (10 uM)
- 4 uL PC DNA / 2.5 uL G DNA / 1 uL I DNA
- 0.5uL KOD Hot Start Polymerase
Total: 25uL
Samples
-------
(3 DNA samples x 3 Primer sets) + NC samples x 2 primer sets
Sample IDs
----------
1. NC, Primers 1
2. NC, Primers 2
3. G, Primers 1
4. G, Primers 2
5. G, PC Primers
6. I, Primers 1
7. I, Primers 2
8. I, PC Primers
9. PC, PC Primers
10. PC, Primers 1
11. PC, Primers 2
Program 2
---------
Activation 95C for 2 min
(
Denature 95C for 20s
Annealing 53C for 10s
Extension 70C for 45s
) x 30 cycles
Program 3
---------
Activation 95C for 2 min
(
Denature 95C for 20s
Annealing 53C for 10s
Extension 70C for 100s
) x 30 cycles
Gel recipe
----------
1 g agarose in 100 mL 1X TBE run at 70V
Loading Samples
---------------
Ladder:
3uL Elution Buffer, 1uL EZ vision 3 dye, 2uL NEB 1kb ladder
Samples:
1uL EZ vision 3 dye, 5uL PCR product
Layout
------
From left to right (with grooved-side of gel facing you). Same layout for both
PCR programs.
Lane Sample ID
~~~~~~~~~~~~~~~~~
L
| .
| Ladder
| 1
| 2
| 3
| 4
| 5
| 6
| 7
| 8
| 9
| 10
| 11
| .
| .
R
Gel Results
-----------
Program 2: No Bands except positive control (including ladder). Think lanes
leaked. Will rerun next time.
Program 3: All samples with control region primers either smeared or had no
bands. My DNA extractions showed a band at ~500bp with the positive control
primers. Curiously the positive control tissue didn't have a band when paired
with positive control primers.
Met with Sarah, she suggested: Decreasing annealing temperature to 49C and
increasing annealing time to 20s. Annealing temperature could be further
lowered to 47 or 45 later. Decreasing cycle number to 25.
Got 4 new positive control samples (GBB, GBH, FBB, FBH) since the previous one
was exhausted.
Nanodrop Concentrations
-----------------------
GBB - 5.2 ng/uL
GBH - 1.5 ng/uL
FBB - 5.8 ng/uL
FBH - 1.0 ng/uL
Next steps: Rerun program 2 gel.
===============================================================================
September 22nd, 2017
===============================================================================
Reran program 2 gel. Same results - No bands except in positive control DNA
with positive control primers.
-------------------------------------------------------------------------------
PCR 5
-------------------------------------------------------------------------------
Made up a PCR master mix with enough for ~ 4 more PCR runs.
Mix 4
-----
Master mix recipe
-----------------
Standard 10x Taq buffer 100uL
MgCl2 60uL
dNTPs 100uL
Hot Start KOD Polymerase 20uL
Made stocks of all DNA samples normalized to approx. 5 ng/uL. 13.2uL of these
samples can be added directly to the PCR samples..
PCR Sample Recipe
-----------------
Master Mix 5.6 uL
Diluted DNA Sample 13.2 uL
Mixed Primers 1.2 uL
Sample IDs
----------
ID # DNA Primers
~~~~~~~~~~~~~~~~~~~~~~~
1 PC PC
2 G PC
3 I PC
4 NC PC
5 PC 1
6 G 1
7 I 1
8 NC 1
9 PC 2
10 G 2
11 I 2
12 NC 2
Program 4
---------
Activation 95C for 2 min
(
Denature 95C for 20s
Annealing 49C for 20s
Extension 70C for 70s
) x 25 cycles
Layout
------
From left to right (with grooved-side of gel facing you).
Lane Sample ID
~~~~~~~~~~~~~~~~~
L
| .
| Ladder
| 1
| 2
| 3
| 4
| 5
| 6
| 7
| 8
| 9
| 10
| 11
| 12
| .
R
===============================================================================
September 25th, 2017
===============================================================================
Gel Results
-----------
No bands. Back to the drawing board. Possible next steps:
1. Redo extractions, the template might be poor quality or sheared. We're
running out anyway.
2. Add DMSO
3. Try regular Taq
4. Increase primer concentrations
Actual plan:
- Revert to pcr 4 program 3. Run some samples with DMSO. Some samples with
original protocol. Some samples with increased primer concentration. Some
samples with regular Taq.
- Switch negative control to a no template control.
===============================================================================
September 27th, 2017
===============================================================================
Asked Maia for PCR advise. She said to definitely use the thermocyclers in the
GGG lab rather than the ones in Schaack lab. They worked much better for her.
Read: Long PCR amplification of the entire mitochondrial genome from individual
helminths for direct sequencing (Hu et al., 2007).
- Advised to definitely use long range PCR.
- Decrease extension time to around 60 degrees due to AT rich region.
Google says that freezing polymerase in master mix ruins it I guess. Will have
to dispose of it, very stupid and wasteful mistake.
Taking another look at the primers I think I'd like to redesign them before
going too much further. They have multiple mismatches and I don't really think
that is necessary. Also having longer primers, smaller product would be nice.
Primer Design 2
---------------
Created a MSA with long and short versions of mtDNA assembly, Chinese sequence
and sequence which Dieter sent over. Used the short assembly produced for IB as
a template. Extracted the first 850bp of ND1 to use as a target for the reverse
primer. Then extracted 850bp of shared sequence at the end of the assembly to
use as the forward primer target.
Used primer3 web interface to generate primers. Specified a minimum primer
length of 21bp, GC content of 40-60%, max 3' self-complementarity=0. Excluded
any primers with mismatches to Dieter's sequence. For the second set of primers
I increased the GC content bounds to 30-70 % to get a smaller expected product
size. Sent to Sarah to check.
===============================================================================
September 28th, 2017
===============================================================================
Redid DNA extractions for a bunch of control Daphnia for use in this and future
experiments. Followed Maia's ammended Zymo extraction protocol as before.
Extracted DNA from five animals from 6 control lines (2 Germany, 2 Israel, 2
Finland). Did two extractions from each line for a total of 12 extractions. For
half of the extractions I flash froze the Daphnia in liquid N2 before
homogenizing with pestle. Sarah suggested that this might help to break down
the carapace.
Extractions all went well - all concentrations ~ 20ng/uL with the exception of
one which is ~6 ng/uL. Stored at 4 degrees. Will aliquot and freeze tomorrow.
===============================================================================
September 29th, 2017
===============================================================================
Sarah asked me to send Hannah some mtDNA assemblies for the G lines. The ones I
have now are in pretty bad shape so I started a fresh run. Created a new
NOVOPlasty seed sequence in
fmacrae/daphnia-mtdna-ma/data/ref/magna/mit/novoplasty_seed.fa. To produce the
seed I took the European reference sequence from Dieter and trimmed off
approximately 2000 bp on either end. This should guarantee that incorrect
sequence won't become the initial seed to the assemblies.
===============================================================================
October 20th, 2017
===============================================================================
Received the new primer sets.
Fwd Primer 3:
ACC GTA TCG AGG TAG CCC TTT
Rev Primer 3:
TCA ATA GTC GAG TTC TTG TCT GG
Expected product size: 600 - 2600 bp
Fwd Primer 4:
GCA GTA ATT TCT AAG AAC ACA ATA TCC
Rev Primer 4:
CCC AAT TTA ATA CTT AAA CCA ACC
Expected product size: 400 - 2400 bp
Made 100 uL 10uM aliquots in TE buffer. Will do some PCR on Monday.
===============================================================================
October 20th, 2017
===============================================================================
-------------------------------------------------------------------------------
PCR 6
-------------------------------------------------------------------------------
Repeat program 3 with the new primers/tissue. Vary extension time as to target
lower and upper bounds of product size.
Use the BIO-RAD T100 thermocyclers in the GGG classroom this time.
Mix 3
-----
- 2 uL 10X Standard Taq buffer
- 1.2 uL 25mM MgCl2
- 2 uL dNTPs (2 mM each)
- 14.2 uL Nuclease Free Water
- 0.6 uL Fwd Primer (10 uM)
- 0.6 uL Rev Primer (10 uM)
- 1 uL DNA Extraction
- 0.4 uL KOD Hot Start Polymerase
Total: 20uL
Master Mix
----------
(Mix x 20)
- 40 uL 10X Standard Taq Buffer
- 24 uL 25mM MgCl2
- 40 uL dNTPs
- 284 uL Nuclease Free Water
- 8 uL KOD Hot Start Polymerase
PCR Sample Recipe
-----------------
- 17.8 uL Master Mix
- 1 uL DNA Extraction
- 0.6 uL Fwd Primer
- 0.6 uL Rev Primer
Samples
-------
((2 DNA samples + NC) x 3 Primer sets) = 9
Sample IDs
----------
1. NC, PC Primers
2. F, PC Primers
3. G, PC Primers
4. NC, Primers 3
5. F, Primers 3
6. I, Primers 3
7. NC, Primers 4
8. F, Primers 4
9. G, Primers 4
Program 5
---------
Activation 95C for 2 min
(
Denature 95C for 20s
Annealing 48C for 20s
Extension 70C for 8s
) x 35 cycles
Program 6
---------
Activation 95C for 2 min
(
Denature 95C for 20s
Annealing 48C for 20s
Extension 70C for 60s
) x 35 cycles
Gel recipe
----------
1 g agarose in 100 mL 1X TBE run at 70V
Loading Samples
---------------
Ladder:
6uL Nanopure water, 2uL EZ vision 3 dye, 4uL NEB 1kb ladder
Samples:
2uL EZ vision 3 dye, 10uL PCR product
Layout
------
From left to right (with grooved-side of gel facing away from you). P(5/6) =
Program 5/6
Lane Sample ID
~~~~~~~~~~~~~~~~~
L
| Ladder
| P5 - 1
| P5 - 2
| P5 - 3
| P5 - 4
| P5 - 5
| P5 - 6
| P5 - 7
| P5 - 8
| P5 - 9
| P6 - 1
| P6 - 2
| P6 - 3
| P6 - 4
| P6 - 5
| P6 - 6
| P6 - 7
| P6 - 8
| P6 - 9
| Ladder
R
Gel Results
-----------
No bands except ladder.
Will try with regular Taq next time.
===============================================================================
October 25th, 2017
===============================================================================
-------------------------------------------------------------------------------
PCR 7
-------------------------------------------------------------------------------
Run PCR with Qiagen Taq PCR Master Mix, and Phusion.
Reduced extension temperature to 60 C as recommended by Su et al., 1996
Added high concentrations of MgCl2 due to recommendations in this thread:
https://www.researchgate.net/post/What_can_I_do_to_have_pcr_products_with_A-T_\
rich_sequences_using_genomic_DNA_as_template
Used BIO-RAD T100 thermocyclers.
Protocols
---------
Taq: Qiagen Quick Start Protocol
Phusion: New England Biolabs
Both saved locally in protocols directory.
Mix 4 (Taq Master Mix)
----------------------
- 12.5 uL Taq Master Mix
- 1.5 uL Fwd Primer
- 1.5 uL Rev Primer
- 2 uL DNA Extraction
- 7.5 uL Nuclease Free Water
Total: 25uL
Mix 5 (Phusion)
---------------
- 5 uL Phusion HF Buffer
- 0.5 uL dNTPs (10 mM)
- 5 uL MgCl2 (25 mM)
- 1.5 uL Fwd Primer (10 uM)
- 1.5 uL Rev Primer (10 uM)
- 2 uL DNA Extraction
- 0.25 uL Phusion polymerase
- 9.25 uL Nuclease Free Water
Total: 25uL
Master Mix (Mix 5)
------------------
(Mix x 10)
- 50 uL Phusion HF Buffer
- 5 uL dNTPs
- 50 uL MgCl2
- 2.5 uL Phusion Polymerase
- 92.5 uL Nuclease free water
Samples
-------
((2 DNA samples + NC) x 3 Primer sets) = 9
Sample IDs
----------
1. NC, PC Primers
2. F, PC Primers
3. G, PC Primers
4. NC, Primers 3
5. F, Primers 3
6. I, Primers 3
7. NC, Primers 4
8. F, Primers 4
9. G, Primers 4
Program 7 (Taq Master Mix)
--------------------------
Activation 94C for 3 min
(
Denature 94C for 10s
Annealing Primers 3: 51C / Primers 4: 46C / PC Primers: 50C for 40s
Extension 60C for 180s
) x 35 cycles
Final Extension 60C for 10 minutes
Primers 3 = Row A (Back)
PC Primers = Row C
Primers 4 = Row H (Front)
Program 8
---------
Activation 98C for 30s
(
Denature 98C for 10s
Annealing Primers 3: 65C / Primers 4: 63C / PC Primers: 59C for 30s
Extension 60C for 180s
) x 35 cycles
Final Extension 60C for 10 minutes
Primers 3 = Row A (Back)
Primers 4 = Row D
PC Primers = Row H (Front)
Primers 3 placed in back row (hotter), primers 4 placed in front row (cooler).
Gel recipe
----------
1 g agarose in 100 mL 1X TBE run at 70V
Loading Samples
---------------
Ladder:
6uL Nanopure water, 2uL EZ vision 3 dye, 4uL NEB 1kb ladder
Samples:
2uL EZ vision 3 dye, 10uL PCR product
Layout
------
From left to right (with grooved-side of gel facing away from you). P(7/8) =
Program 7/8
Lane Sample ID
~~~~~~~~~~~~~~~~~
L
| Ladder
| P7 - 1
| P7 - 2
| P7 - 3
| P7 - 4
| P7 - 5
| P7 - 6
| P7 - 7
| P7 - 8
| P7 - 9
| P8 - 1
| P8 - 2
| P8 - 3
| P8 - 4
| P8 - 5
| P8 - 6
| P8 - 7
| P8 - 8
| P8 - 9
| Ladder
R
===============================================================================
October 26th, 2017
===============================================================================
Gel Results
-----------
No bands in negative controls.
Positive controls primers: A clear band at <1kb for both polymerases and both
DNA samples. Some extra bands visible >1kb in the F sample with phusion
polymerase. Probably nonspecific amplification.
Primers 3: Clear bands visible at <1kb for both polymerases both tissues. In
phusion G sample a clear band is visible at ~1.7kb
Met with Sarah:
- We agreed that the extra bands in the phusion samples are probably artifacts
from nonspecific contamination.
- This supports the existing genome sequence for D. magna from Dieter, but
doesn't solve our problem with artifacts at the ends of the alignments.
- We decided to go ahead and sequence using primer pair 3.
===============================================================================