Team:LASATX/Notebook

Notebook

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Document the dates you worked on your project.

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Chronological notes of what your team is doing.
Brief descriptions of daily important events.
Pictures of your progress.
Mention who participated in what task.

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6/2 Pia, Dylan, Caleb

Reactants
Hotstart taq PCR supermix (platinum) 45 uL
→ instead of platinum, use Hotstart Accuprime pfx mastermix (recombinant DNA polymerase)
DNA template* (10 ng) 1 uL
primers (20 uM each) 1 uL x 2
water 2 uL
Total Vol 50 uL
*use the 1:10 dilution (clear tube, yellow sticker)
1.5 Gel electrophoresis of PCR product (1000 kb) to make sure DNA actually there/rxn works.
PCR amplifying cowg4 with new cowg4 primers; PCR amplify PCR 2.1 vector (TOPO-TA cloning vector) with vector primers (gibsoning fragment into TOPO-TA vector)
Touchdown PCR
Thermocycler
95 C 2 min (if Hotstart, then 5 min) (denature)
// 10 cycles //
95 C 20 s (denature)
0.3 C/s to 50 C (anneal)
72 C X s (extension = 1 min/kb)

// 15 cycles //
95 C 20 s
55 C 20 s
72 C 1 min

72 C 10 min
4 C forever

Made:
COWg4 tubes: in thermocycler next to window
purple: in block A; COWg4 fragment using regular accuprime protocol
green: in block B; COWg4 fragment using touchdown protocol (above)
Vector 2.1: in thermocycler across from Michelle’s (the usual one), block B using CA protocol

pink: pcr 2.1
yellow: pcr 2.1

6/3/15 Isaree

Made gels (1% TAE)

Made two gels; used large combs

100 mL TAE buffer + 1g agarose (Nuseive 3:1)
stir
microwave
add 1 drop EtBr
let cool

Gel electrophoresis: Ran pcr products (COWg4 and vector 2.1) through gels

COWg4: wells on left
1) 1 kb ladder (2uL), loading dye (2uL), water (10 uL)
3) purple tube (frag4, reg accuprime) (30uL), dye (5 uL)
5) teal tube (frag4, touchdown) (45 uL), dye (7.5 uL)

Result: FAILED
nothing showed up at 1 kb line, everything smaller than 50 kb…?

PCR 2.1: wells on left
1) 1 kb ladder (2uL), loading dye (2uL), water (10 uL)
3) pink tube (2.1) (40 uL), dye (6.7 uL)
5) yellow tube (2.1) (45 uL), dye (7.5 uL)

Result: PASSED??
both products (3 and 5) showed up at exactly 1 kb line
hypothesis: tubes are actually of COWg4, but were mislabeled when put in thermocycler??
pink (3) and yellow (5) tubes, labeled “2.1”, both run using Touchdown PCR protocol, actually contained COWg4??

Gel purification: purifying mislabeled COWg4 tubes

3: 0.151 g
5: 0.090 g

Dissolving the Gel Slice
1. Weighed 1.5 mL tube.
2. Added MBS. Accidentally added 15.1 uL to 3 (instead of 151) and 9 uL to 5 (instead of 90). Vortexed/incubated 10 min at
50C. Then added and extra 300 uL to each tube. Incubated at 50C for another 10 min.

DNA Purification by Centrifugation
1. Place one SV Minicolumn in a Collection Tube for each dissolved gel slice or PCR amplification.
2. Transfer the dissolved gel mixture or prepared PCR product to the SV Minicolumn assembly and incubate for 1 minute at RT
3. Centrifuge at 16,000 × g for 1 minute, and discard liquid
4. Wash with 700µl of Membrane Wash Solution. Centrifuge 1 minute at 16,000 × g, discard. Wash with 500µl of Membrane Wash
Solution, centrifuge 5 minutes at 16,000 × g.
5. Remove, discard, recentrifuge 1 minute with the microcentrifuge lid open (or off ) to allow evaporation of any residual
ethanol.
6. Transfer SV Minicolumn to a clean 1.5ml microcentrifuge tube. Apply 50µl of Nuclease-Free Water directly to the center of
the column. Incubate at RT for 1 minute. Centrifuge 1 minute at 16,000 × g.
7. Discard SV Minicolumn.

4. Nanodrop
nanodropped, got around 5 ng/uL
very low probably b/c eluted with 50 uL nuclease-free water
~SpeedVac for 20 min (run, auto)~
nanodropped again, still got around 5 ng/uL

Stored tubes (3 and 5) containing the eluted DNA (COWg4) at –20°C in NW corner of Synbio Box #3.

NEXT TIME:

speed vac/nanodrop tubes 3 and 5 (from synbio box #3), until they reach around 20 ng/uL, but leave at least 10 uL in the tube. gibson COWg4 fragment into pcr 2.1 vector, using fragment DNA from tube with higher concentration (either tube 3 or 5), and using the pcr 2.1 vector from the pink pcr tube labeled “pcr 2.1” on side, in NW corner of Synth Bio PCR Tube box.

6/3/15 Isaree, Anna, Pia, Sam

speed vac/nanodrop tubes 3 and 5 to around 50 ng/uL

3: 52.1 ng/uL
5: 53.1 ng/uL

re-did pcr reaction for COWg4 (to gibson into pcr 2.1)

used mb_COWG4_PCR2_1 primers
made 20uM stock of primers (SynBio box #3)
AccuPrime pfx supermix (from Pia, need to order more)
touchdown pcr: problem: annealing temp too low (lower than Tm)

gibsoned COWg4 (tube 5) into pcr 2.1

gibson mix, 1.3 X (15uL)
insert (.94 uL COWg4, 1.56 uL water)
vector (2.5 uL)
total: 20 uL
60 min at 50 C
stored at: XXC

ran re-do of COWg4 pcr through gel

ladder + 40 uL COWg4
product all below 50 bp
hypothesis: tubes were labeled correctly; gibsoned reaction was (some form of) vector 2.1 into vector 2.1

figured out how to design primers on geneious; more parameters

homo- and hetero-dimers
delta G: favorability of reaction
expression primers for COWg 1, 2, 3, 4 (putting fragments into pET21 expression vector)
working on primers to insert COWg 1, 2, 3, 4 into pcr 2.1 vector
purification of failed COWg4 pcr 2.1 (Anusha, James, Jasmine)

Need to do: (by the end of today)

order more accuPrime
→ so that we can redo pcr of COWg4 with lower annealing temperature (should be 5 degrees below Tm)
order COWg primers for gibsoning insert into pcr 2.1 vector

6/9/15 Isaree, Pia, Anusha, Sam

if check the relative delta G/free energies, much more negative (so much more likely) for complete complementary binding
Tm for PCR - ALWAYS what anneals in first cycle
if check Tm of hairpins, much LOWER than tm of primer, so hairpins likely to melt apart

First pcr of COWg4 (Pia)

taq
2 ul (24 ng)
Touchdown protocol

Second pcr of Cowg4 (Isaree)

pfx (aliquot from Michelle)
3 uL (36 ng) template
2-step PCR (allows less secondary structure since higher temp)
added 1mM MgCl2 (stabilize primer binding)

Reactants

Hotstart AccuPrimer pfx 45 uL
COWg4 template (10 ng) 3 uL
primers (20 uM each) 1 ul x2
MgCl2 (100 mM) 0.5 uL

2-step PCR Protocol
95C 5 min (Hotstart)
///25 cycles///
95C 20s
68C 1 min
///////////////////
72C 10 min

oh wait the primer dilutions are wrong
nanodropped:
COWg_2.1 F = 59 uM
COWg_2.1 R = 58 uM
diluted to 20 uM for both

3. Third pcr of COWg4

20 uM primers, pfx, 3 uL (~ 30 ng) template, MgCl2, 2step protocol

4. Fourth pcr of COWg4

20 uM primers, pfx, 3 ul, MgCl2, 3step (touchdown) protocol

5. designed primers for:

gibsoning fragments into pET21 expression

COWg 1, 2, 3, 4
vector primers

gibsoning fragments into individual cloning (pcr 2.1) vectors

6/10/2015 Isaree, Pia, James, Anusha

1. speedvac/nanodrop COWg4 A and B

higher concentration in A (touchdown protocol) vs B (2 step)

2. gibson COWg4 A into pcr 2.1

3. First chemical transformation: 4, 2.1, TOP10 “bad”

heat shocked cells, then realized SOC media was contaminated
left in ice ~2 h after heat shock

4. Second chemical transformation: 4 “good”

5. Assigned lab jobs