Team:USP-Brazil/GaTE

GaTE Lab - Lab Diary

22 May – 24 May
Plasmid BBa_E1010 that contains RFP gene was transformed into DH10B and plated on kanamicin plates from which a single colony was inoculated for miniprep.
Plasmid extraction successful was detected by running an agarose gel and its quantification was determined by nanodrop.
RFP gene was amplified by Gradient PCR for further assembly. It was performed using 1/10 and 1/100 dilutions of a RFP sample 1540 ng/uL.
1/100 dilution at 65°C appears like optimal PCR conditions for RFP PCR.

3 Jun – 7 Jun
To verify in which annealing temperature the pAOX1 promoter right size fragment is obtained a Gradient PCR was performed. pAOX1 amplification was executed by using GoTaq Polimerase
PCR was performed with pAOX1 primers dilution from 100 mM (correspondent to 1/10 dilution from stock) to 10 mM (correspondent to 1/100 dilution from stock), 3 different yeast gDNA concentration (750 ng/uL,1606 ng/uL and 1303 ng/uL) and in a 53°C to 65°C temperature range.
Best conditions were detected by analysing Gradient PCR on a gel run: 1/100 dilution at 53°C  appears most reliable, but 1/10 dilution is most pronounced

10 Jun – 14 Jun
pAOX1 was amplified with less quantity of Q5 High Fidelity 2X Master Mix and longer thermocycling extension to compare to 24/5/13 run using 1/100 dilution of RFP template
PCR conditions tested for Q5 High Fidelity 2X Master Mix:

• annealing temperature of 53°C for pAOX1 (7/06/13 gradient PCR) along with the use of 1/10 yeast gDNA dilution
• 67°C annealing  temperature and 0.5 uL of 1/100 dilution RFP template

Found out best conditions to amplify pAOX1 (which are shown in the end of the lab diary)
pPIC9K digest with EcoRI and NotI and kept frozen
Gibson assembly using NEB Gibson Assembly cloning kit of pAOX1, RFP and EcoRI/NotI digested pPIC9K
Transformation of DH10B E. coli with pPIC9K using electroporation
Select, inoculate, and incubate colonies from Gibson plates (pPIC9K-pAOX1-RFP) and pPIC9K plates
Plasmid extraction of transformed colonies

17 Jun – 21 Jun
Transformation of DH10B and NEB 5-alpha competent E. coli with Gibson assembly product using electroporation and heat shock, respectively
Plate transformed cells
Select, inoculate, and incubate colonies from heat shock and electroporation Gibson assembly plates
Plasmid extraction and plasmid digestion gel (Gibson assembly with NotI enzyme)
RFP PCR amplified at 69 °C annealing temperature

28 Jun – 30 Jun
RFP PCR using new Gibson assembly compatible primers
DNA gel to check
Gibson assembly of pAOX1, RFP, and EcoRI/NotI digested pPIC9K and transformation using NEB 5-alpha competent cells (heat shock) and DH10B (electroporation), plating 100 uL and 900 uL for each on ampicillin plates
Select, inoculate, and incubate from plates
Spin down and freeze samples

1 Jul – 5 Jul
Plasmid extraction from inoculated colonies
Plasmid digestion using NotI
DNA agarose gel to check. Plasmid digestion of heatshock transformed samples
PCR of pAOX1 and RFP followed by purification using NucleoSpin PCR Clean-up kit
Gel of PCR products and digests to check
Quantification of RFP PCR product and pPIC9K previously digested by using gel and Nanodrop

	Quantification by Nanodrop	Quantification using gel
RFP	104 ng/ul	8 ng/uL
pPIC9K	141 ng/uL	140 ng/uL

NucleoSpin Clean-up of pPIC9K overnight digest
DNA gel of pAOX1 PCR and pPIC9K digest purification
Repeat pAOX1 PCR to obtain more pAOX1 sample
Combine pAOX1 PCR products and purify with NucleoSpin Clean-up
DNA gel of purified pAOX1 and pPIC9K digested/undigested
pAOX1 PCR using 2 uL of 1/10 yeast genomic DNA with 50°C and 57°C annealing temperatures
Overnight digest of pPIC9K
pAOX1 PCR at 60°C annealing temperature
DNA gel of 60°C annealing PCR product; 5 uL PCR product and 2 uL loading buffer
Pool both paOX1 PCR products together and run an agarose gel
Run pPIC9K overnight digestion on gel
Gel extraction of pAOX1 and pPIC9K

8 Jul – 12 Jul
Gibson assembly of pAOX1, RFP, and pPIC9K
Results: no colony formation on plates
Transformation using more Gibson assembly product next time
Overnight digest of pPIC9K with EcoRI and NotI
Gel purification of pPIC9K digestion
DNA gel of pPIC9K

15 Jul – 16 Jul
Gibson assembly of pPIC9K, pAOX1, and RFP
Retransform Gibson assembly reaction mixture
Result: no colonies

24 Jul – 26 Jul
Digest of pPIC9K with BamHI and NotI
Run agarose gel and gel extraction purification of digested pPIC9K
Pull together the two purified and digested pPIC9K samples and run a gel
RFP PCR (using new primer (oldschool_F_RFP)
RFP PCR product run on agarose gel for gel extraction purification
pJET blunt ligation of RFP PCR product
pJET-RFP transformation

29 Jul – 3 Aug
Miniprep of pJET-RFP
Overnight digestion of pJET-RFP using BamHI and NotI
Gel extraction purification of RFP
DNA gel to get RFP concentration
Ligation of pPIC9K and RFP overnight
Transform ligation reaction mixture using electroporation
Remove salt from 30/7/13 pPIC9K-RFP ligation
Transformation of DH10B electrocompetent E. coli with salt-removed pPIC9K-RFP

Ligation of pPIC9K and RFP
Inoculate pPIC9K from glycerol stock
Heat inactivation of T4 ligase from pPIC9K and RFP ligation
Remove salt from ligation sample
Transformation of “purified” pPIC9K-RFP with electrocompetent DH10B
Inoculation and miniprep of pPIC9K
Digestions of pPIC9K with BamHI, NotI, and BglII (also RFP, pSB1C3 with EcoRI)
Transformation of Life Technologies synthesized plasmids pAOXmod_RFPmod and Mxr1p_minimum

4 Aug – 9 Aug
RFP (from previous week) digestion with EcoRI; result: just use new pSB1C3 glycerol inoculation
Gel extraction purification of pPIC9K digestion. Result: smear
Life Technologies samples: Inoculation of samples as well as pSB1C3
pPIC9K overnight digestion with less enzyme than last digestion
Repeat pPIC9K digestion because mixture didn’t have proper amount of sample
Quantify by gel
Ligation of RFP and pPIC9K
Inoculation of pPIC9K for miniprep to get more sample
Inoculation of pAOX_RFP and Mxr1p for glycerol stock
Life Technologies samples miniprep (pAOX_RFP, Mxr1p, pSB1C3)
Pool pAOX_RFP samples together. Pool Mxr1p samples together and then also pSB1C3 samples
Digestion of pAOX-RFP, Mxr1p, and pSB1C3 for quantification

• pAOX_RFP with NotI (50 ng/uL)
• Mxr1p with XbaI (50 ng/uL)
• pSB1C3 with XbaI (20 ng/uL)

Digestion of pAOX_RFP with EcoRI and NotI, and also another with EcoRI and SpeI
Transformation of pPIC9K-RFP ligation of this week
Verification gel of pAOX_RFP digestion before doing gel extraction purification
Gel extraction purification of pAOX_RFP digestions (EcoRI/NotI and EcoRI/SpeI)
Miniprep of pPIC9K
Pick pPIC9K-RFP colonies and inoculate
pPIC9K digestion with EcoRI for quantification
pPIC9K, pSB1C3, and Mxr1p (all this week samples) digestions

• pPIC9K with EcoRI for quantification
• pSB1C3 with EcoRI and SpeI
• Mxr1’p with EcoRI and PstI

Low pPIC9K concentration, so prepare another inoculation for miniprep
Gel extraction purification of pSB1C3 and Mxr1p samples
Digestion of remaining pPIC9K sample with EcoRI and NotI
Life Technologies samples: miniprep of pPIC9K_RFP
Repeat pSB1C3 digestion (EcoRI and SpeI) and Mxr1p (EcoRI and PstI)
Gel verification of pSB1C3, Mxr1p, and pPIC9K digestions
pSB1C3 digestions looks complete
Add more enzyme to Mxr1p and pPIC9K
Gel extraction purification of pPIC9K and pSB1C3
Miniprep of pPIC9K
Life Technologies samples: Gel of pPIC9K_RFP digestion with KpnI and run a gel - some lanes look like proper pPIC9K_RFP plasmid samples

12 Aug – 17 Aug
Quantification of pPIC9K miniprep sample from last week (16 ng/uL)
Use speedy-vac to increase the sample concentration, new concentration: 101.3 ng/uL
pPIC9K digestion with EcoRI and NotI Gel extraction purification of Mxr1p digested with EcoRI and NotI and pPIC9K digested with EcoRI and NotI
pSB1C3 digestions with EcoRI/SpeI  and EcoRI/PstI
pSB1C3 inoculations
Quantification pPIC9K-RFP
pPIC9K digestion with EcoRI and NotI
Gel extraction purification of: pSB1C3 (digested by EcoRI/SpeI and EcoRI/PstI) and pPIc9K (digested with EcoRI/NotI)
Pellet pSB1C3 inoculations
Gel purification continued (NTI buffer overnight)
Miniprep of pSB1C3 samples, but a mistake was done which resulted in lower yield
Ligation of pSB1C3 (15/8/13) with pAOX_RFP
Ligation of pSB1C3 (15/8/13) and Mxr1p
Plate ligations on plates with chloramphenicol
Quantification of pSB1C3 by digestion and gel
Inoculation of pAOX_RFP-pSB1C3, Mxr1p-pSB1C3 and pPIC9K
Inoculation of pPIC9K for glycerol stocks
Pellet pAOX_RFP-pSB1C3, Mxr1p-pSB1C3 and pPIC9K
Make glycerol stock for pPIC9K

19 Aug – 24 Aug
Miniprep of pAOX_RFP-pSB1C3, Mxr1p-pSB1C3 and pPIC9K
Verification of pAOX_RFP-pSB1C3 and Mxr1p-pSB1C3

• pAOX_RFP-pSB1C3 HindIII and EcoRI (each cut once, gel should show 2.8 kb and 800 bp bands)
• Mxr1p-pSB1C3 with BsaI and PstI (should show 1 kb and 2 kb bands)

Concentrate pSB1C3 miniprep last week sample (from 5 ng/uL to 19.4 ng/uL)
pAOX_RFP-pSB1C3 digest verification to free the insert using EcoRI and SpeI
Pool pPIC9K miniprep samples together and quantify (50 ng/uL)
DNA gel with pAOX_RFP-pSB1C3 samples and also, undigested Mxr1p and pPIC9K quantification
Mxr1p-pSB1C3 (from last week) transformation
pAOX_RFP-pSB1C3 transformation
Inoculation of Mxr1p-pSB1C3, pAOX_RFP-pSB1C3, and Mxr1p from Life Technologies
Pellet inoculations

26 Aug – 30 Aug
Miniprep of last week Mxr1p-pSB1C3 samples
Mxr1p-pSB1C3 digestion verification with BsaI and PstI
Pool pAOX_RFP-pSB1C3 together and Mxr1p from Life Technologies samples together
Digestion verification of pAOX_RFP-pSB1C3 and Mxr1p from Life Technologies

• pAOX_RFP-pSB1C3  with HindIII and EcoRI
• Mxr1p with EcoRI and NotI

Mxr1p-pSB1C3 digestion verification with EcoRI/PstI
Transform pAOX_RFP-pSB1C3 and Mxr1p-pSB1C3 for glycerol stocks
Prepare glycerol stocks by inoculation glass flasks from plates
Quantification of pAOX_RFP-pSB1C3 and Mxr1p (from Life Technologies) from this week (45 ng/uL)
Finish making glycerol stocks for pAOX_RFP-pSB1C3 and Mxr1p-pSB1C3 samples
Inoculation of Mxr1p-pSB1C3 for miniprep
Miniprep of Mxr1p-pSB1C3
Digestion quantification of Mxr1p-pSB1C3 miniprep (not quantifiable because there wasn’t undigested sample)

2 Sep – 6 Sep
Mxr1p-pSB1C3 last week sample quantification (140 ng/uL)
pPIC9K digestion with BamHI and NotI
PCR of RFPmod from pAOXmod_RFPmod-pSB1C3
DNA gel verification of digestion and PCR

• Digestion showed both 9kb and 300bp bands
• PCR appeared to have some unspecific bands

DNA gel extraction purification
DNA gel of pPIC9K (BamHI/NotI digested) and RFPmod
pJET blunt ligation of RFPmod
pJET ligation product transformation by electroporation

9 Sep – 12 Sep
Inoculation of pJET-RFPmod samples
Transformation of pAOXmod_RFPmod_pPIC9K
Miniprep of pJET-RFPmod samples
Digestion of pJET-RFPmod samples
Inoculation of 2 pAOXmod_RFPmod_pPIC9K colonies
Gel extraction purification of RFPmod
PCR of RFPmod (for pSB1C3) [4/9/13]
PCR of pAOXmod (for pSB1C3) [3/7/13]
PCR of RFPmod for pFLD
Gel to quantify RFPmod (BamHI/NotI) purified by gel extraction (7.5 ng/uL)
Ligation of pPIC9K (from last week) and RFPmod (from this week) (both digested with BamHI/NotI)
Pellet pAOXmod_RFPmod_pPIC9K samples for miniprep
Gel extraction purification of RFPmod, pAOXmod, and RFP-pFLD PCR
pJET blunt ligation of these purified PCR products
Miniprep of pAOXmod_RFPmod_pPIC9K samples

Running summary of best reaction conditions:

pAOX1 PCR  (3/7/13)

	volume (uL)
1/10 dilution of yeast genomic DNA	2
pAOX1 fwd primer	0.5
pAOX1 rv primer	0.5
water	3.25
Q5 2X Master Mix	6.25
total	12.5

RFP PCR (20/6/13)

	volume (uL)
1/100 RFP template	0.5 (7.7 ng)
Fwd RFP primer	0.5
Rv RFP primer	0.5
water	4.75
Q5 2X Master Mix	6.25
total	12.5

pPIC9K digestion with EcoRI and NotI (overnight) (3/7/13)

	volume (uL)
pPIC9K (~140 ng/uL)	20
NEB EcoRI	0.5
NEB NotI	1
NEB Buffer 3	3.5
BSA	0.55
water	9.65

For more information, check our Results.