Team:Brown/Notebook weekly Logs/Weekly Team2 Notebook

=Histamine Sensor Weekly Lab Log=

Week 6

July 20, 09

Plan for the week:
 * Run gels: digest OmpC, TetA
 * gel purify
 * ligate Tet into pGEMT-easy→ DH5α transformation
 * ligate OmpC-TetA standard assembly→ DH5α transformation
 * OmpR BB: grow cultures tonight→ genomic purification tomorrow
 * PCR Taz1BB
 * Amp-Kan-Tet plates
 * Mutagenic PCR

Tasks accomplished today:

1) Ashley did PCR of Taz1 (i) Resuspend dry primers to 100 μM stock (ii) Make 20 μM working stock ( 1μL 100 μM stock + 4 μL H2O= 5 μL total) (iii) Template: Taz1 plasmid (miniprep)
 * BioBrick Primers: Tar BB For (19.3 nm); EnvZ Rev (17.1 nm)
 * Control Primers: Amp Taz 1 For  (23.0 nm); Control Taz1 Rev (26.9 nm)


 * Mastermix: 47 μL
 * Primer For + Rev: 1 μL each
 * 3 tubes of BioBrick, 3 tubes of control

1) 94°C for 5 min 2) 94°C for 30 sec 3) 57°C* for 30 sec 4) 72°C for 1.5 min 5) GoTo 2, 34 times 6) 72°C for 5 min 7) 4°C forever
 * PCR program:

2) Gel results for digests:


 * 1% gel


 * L1: 1 kb ladder
 * L 2: Tet Steph (EcoRI, XbaI)
 * L3: Tet MC ( E,X)
 * L4: Tet Ash (E,X)
 * L5: Tet Steph (E,P)
 * L6: Tet MC (E,P)
 * L7: Tet Ash (E,P)
 * L8: Tet old (E,P)


 * 2% gel


 * L1: 1 kb
 * L2: Omp2 MC
 * L3: Omp1 Steph
 * L4 : Omp 2 Steph
 * L5: 100 bp ladder
 * L6: 100 bp ladder


 * Did gel extraction on all samples

3) Making competent RU1012

(i) Plated RU1012 agar stab on LB-Kan plate (ii) 11 am: started liquid culture (iii) 6 pm: inoculate into SOB broth (2 mL, 4 mL, and 10 mL samples) (iv) check OD600 with 3 μL sample on nanodrop


 * 11:40 am:
 * 2 mL: 0.356
 * 4 mL: 0.380
 * 10 mL; 0.388

4) Testing transformation efficiency of DH5α (i) add 50 μL competent cells + 1 μL plasmid (OmpR miniprep 7a) (ii) incubate 30 mins on ice (iii) heat shock 30 sec at 42 °C (iv) incubate on ice for 2 min (v) 200 μL  LB media (vi) incubate 20 min at 37°C (vii) plate different densities: 100 μL, 10 μL, 1 μL, incubate at 37°C at 4:30 pm (viii) calculate CFU

5) Gel for Taz1


 * Lane 1: 1 kb ladder
 * Lane 2: 100 bp ladder
 * Lane 3: Taz 1
 * Lane 4: Taz 2

July 21, 09

Tasks for today:
 * Run digest again, test E and S on tet
 * Genomic purification of DH5α then PCR OmpR, Run gel
 * PCR Taz1, Run gels
 * Calculate competency of gels

1) Digest of Tet (from 7/16 minipreps) with EcoRI and SpeI


 * Concentration (ng/μL ), μL DNA, μL water
 * Tet Steph: 129.3, 7.737, 8.27
 * Tet Ash: 126.1, 7.93, 8.07
 * Tet Michael: 71.8, 13.93, 2.07

Incubate till 12:00 pm (1.5 hours)


 * Lane1: 1 kb ladder
 * Lane 2: Ash
 * Lane 3: Ash 2
 * Lane 4: 1 kb ladder
 * Lane 5: Steph
 * Lane 6: Steph 2
 * Lane 7: Michael
 * Lane 8: 1kb


 * E, S cut successfully for Tet→ enzymes are ok

2) Genomic purification performed.
 * Primers: OmpR BB Reverse, OmpR BB Forward
 * Mastermix: 47 μL; Primers: 1 each, DNA: 1 μL


 * Lane1: 1 kb ladder
 * Lane 2: 1 kb ladder
 * Lane 3: Tube 1
 * Lane 4: Tube 2
 * Lane 5: Tube 3
 * Lane 6: Tube 4
 * Lane 7: 100 bp ladder
 * Lane 8: 1kb


 * Do not use tubes 2 and 3 of genomic DNA

3) PCR of Taz 1


 * A: amplification. B: Biobrick; Numbers correspond to Taz samples from minipreps of 7/17)


 * Gel 1
 * Lane 1: 1kb
 * Lane 2: 1A
 * Lane 3: 1B
 * Lane 4: 2A
 * Lane 5: 2B
 * Lane 6: 3A
 * Lane 7: 3B
 * Lane 8: 1 kb


 * Gel 2:
 * Lane 1: 1kb
 * Lane 2: 4A
 * Lane 3: 4B
 * Lane 4: 5A
 * Lane 5: 5B
 * Lane 6: 6A
 * Lane 7: 6B
 * Lane 8: 1 kb

July 22, 09

1) OmpC: Grow liquid cultures, make glycerol stocks, digest with EcoRI and SpeI, run 20 μL on 3% gel with minimal loading dye

2) Sequencing: OmpR, Registry (purified plasmid form), purifying PCR product

3) Sequencing, Registry Taz1→ miniprep transformation in DH5α; make glycerol stocks; purify PCR


 * Purification of OmpR1, Taz 2B, Taz 4A
 * Nanodrop concentrations:
 * OmpR1: 28.1 ng/μL
 * Taz 2B: 16.6 ng/μL
 * Taz 4A: 16 ng/μL


 * Sent in for sequencing

4) tetR: ligation into pGEM T easy (EcoRI, PstI)→ ligation into appropriate expression vector


 * Digest: 8μL pGEM, 8 μL water, 2 μL multicore buffer, 1 μL EcoRI, 1 μL PstI
 * Incubate at 37°C


 * Ligation of TetA into pGEM T-easy
 * 10x buffer 2μL
 * ligase 1 μL
 * vector 1μL
 * DNA 10 μL
 * Water 6 μL
 * Incubate overnight at 4°C

July 23, 09

1) 9am: stop inoculation of ompC, miniprep, nanodrop, sequencing, glycerol stocks 2) Find expression vector for tetR and ligate (from pGEM T-easy-tetR ligation) 3) Design primers for ompC 4) Make RU1012 competent

Taking concentrations of RU1012 00 λ600
 * 10:30 am 0.01
 * 11:30 am 0.03
 * 12:30 pm 0.05
 * 1:10 pm 0.09
 * 2:00 pm 0.12
 * 2:40 pm 0.15
 * 3:10 pm 0.2
 * 3:50 pm 0.26
 * 4:20 pm 0.33
 * 4:50 pm 0.38
 * 5:20 pm 0.50


 * Completed Inouye protocol, stored at -80°C

July 24, 09

1) Inoculated tet-pGEM T-easy in liquid cultures→ miniprep, nanodrop, digest→ expression vector 2) Run gel:
 * Omp: cut with S, P
 * Tet: cut with X,P
 * Plasmid: 2079 bp
 * Tet: 1191 bp
 * OmpC: 108 bp


 * Supercoiled v.s Linear v.s Circular plasmids
 * Supercoiled: naturally produced by E.coli (i.e. miniprep) runs faster than linear plasmid
 * Linear: plasmid fom restriction digest
 * Circular: covalently closed plasmid (i.e ligation) runs slower than linear plasmid

July 25, 09

1) Standard assembly
 * 10 μL Ligation mix

2) Gel Extraction 3) Nanodrop Concentrations:
 * OmpC1: 1.8 ng/μL
 * OmpC2: 3.9 ng/μL
 * Tet3: 12.3 ng/μL
 * Tet1: 4.6 ng/μL

4) Digest OmpC with SpeI and PstI
 * Nanodrop concentration:
 * Omp1 MC: 30.8 ng/μL
 * Omp2 MC: 31.1 ng/μL

5) Ligate Tet3 into OmpC digest 6) Transformation of OmpC-TetA into DH5α and RU1012

Syzmanski Protocol
 * 50 μL cells and 1 μLDNA→ ice for 30 mins
 * Heat shock 42°C for 30 sec
 * 2 min on ice
 * 200 μL LB
 * incubate 20 min at 37°C
 * plate on Amp plates

July 26, 09

1) Inoculated ompC-tetA ligations @ 9:30 am→ miniprep liquid cultures→ glycerol stocks

Week 7

July 27, 09

1) Digests: RBS (SpeI, PstI), TetA (XbaI, PstI), OmpC (SpeI,PstI), TetA-pGEM (XbaI, PstI) 2) Ligation with Digested Tet from July 24th 3) Transform ligation: RBS-tetA→ inoculate ligation→miniprep, glycerol stocks 4) Ran gel: tet-pGEM (digest with X< P); should see 2 bands at 3000 and 1000 bp→ gel purify tetA 5) Digest: pBluscript (160.7 ng/μL)--? Transform into DH5α→ tet plates

July 29, 09

1) Digest SK with X, P 2) Obtain Tet X,P→ ligate into SK
 * sequencing
 * ligation with RBS (done with optimized protocol and standard protocol, incubated at 4°C overnight

3) Make amp-tet plates 4) Transform RU1012 with Taz1 (incubation started at 4:40 pm)→ run SDS page gel

August 2, 09

1) IPTG induction of Taz1 from RU1012
 * 8:30 pm: started liquid cultures (5 mL LB, 5 μL Amp, colony of RU1012+Taz
 * 10:30 am: move 0.5 mL liquid culture to 5 mL new liquid culture
 * IPTG volumes: 5 μL, 6 μL, 7.5 μL, 10 μL
 * 12:30 pm: spun 1 mL aliquot of 6 μL and 7.5 μL liquid cultures, spun 5 mL aliquot of 5 and 10 μL liquid cultures→ freezer
 * 1mL cultures
 * resuspend in 200 μL dH2O
 * take 20 μL of dH2O and move to another tube
 * add 2x sample buffer
 * incubate 5 min at 95 °C, vortex, lyse
 * spin at high speed, load supernatant

Week 8

August 4, 09

1) Tested Mutagenic Primers 2) SDS Page of Taz1→ run for 90 min at 121 V, stain overnight 3) DNA purification of PCR TazMut 4) Ligation of purified PCR product: mut Taz into pGEM

August 5, 09

1) Ligation of OmpR BB and Taz1 BB into pGEM T-easy 2) Transformation into DH5α, plate on Amp plates
 * ligation was unsuccessful→ redo

August 6, 09

1) Redo transformations
 * Control: weird, clear colonies
 * Taz MutC: 2 colonies
 * Taz MutE: some clear colonies
 * Taz MutD: good plate
 * Taz MutA: none
 * Taz Mut B: some
 * Taz BB: some
 * OmpR BB: good plate

2) liquid cultures of Taz B,C,D,E, Taz BB and OmpRBB 3) miniprepped all but TazE (nothing grew) 4) Sent in mutagenic products for sequencing

Week 9

August 10,09

1)	Digests a.	pGEm-OmpR with E,P→ insert b.	pGEM-Taz with E,P-→ insert c.	July 11 miniprep samples of OmpC to get BB ector with E,P cut sites→ vector backbone d.	Ran a gel→ gel extraction e.	Revived glycerol stocks of Taz1 f.	Inoculated July 27 Taz1-DH5α transformation colonies in liquid cultures

August 11, 09

1) Round the Horn PCR!

Primers: (i) P (phosphorylated)-For Mut R1 Taz 1+ Rev Mut R1Taz1-P (ii) P-For Mut R2 Tot Taz1+ Rev Mut R2 Tot Taz1-P (iii) For Mut R2 A Taz1+ Rev Mut R2 Tot Taz1-P (iv) P-For Mut R2B Taz1+ Rev Mut R2B Taz1-P (v) ForMut R1 Taz1+ Rev Mut R1 Taz1 (linear) (vi) For Mut R2B Taz1+ Rev Mut R 2B Taz1 (linear) Primer Phosphorylation: (i) 37 μL H2O (ii) 5 μL PNK buffer (iii) 1 μL 50mM MgSO4 (iv) 5 μL primers (100 μM) (v) incubate at 37°C (vi) kill PNK- heat mixture at 95°C for 5 min

August 12, 09

1) started PCR

PCR mixture
 * 39 μL dH2O
 * 5 μL 10x polymerase buffer
 * 1.5 μL forward primer
 * 1.5 μL backward primer
 * 1 μL DNTPs
 * 1 μL template=miniprep Taz1
 * 1 μL Pfx platinum polymerase

PCR program (i) 95°C 1 min (ii) 93 °C 30 sec (iii) 48 °C 30 sec (iv) 72 °C 18 sec (v) Go to 2 25 times (vi) 4 °C hold

2) Loren Looger’s suggestions for changing Tar to become histidine receptor
 * R69E, R69D, R69Q, R69N
 * R73E, R73D, R73Q, R73N

August 13, 09


 * Ligation of PCR products

August 14, 09

1) PCR amplification of Taz with new primers 2) Gel visualization (bright bands!) 3) Gel extraction (Qiagen) 4) Ligation

August 15, 09

1) Transformation results: no colonies on all except Taz1 PCR (150 μL ) and Taz Gel (50 μL )→liquid cultures 2) Ligation again with different optimized combinations→ good results

August 16, 09

1) miniprep of Taz-pGEM ligations 2) Transformation of Taz-pGEm ligations

Week 10

August 17, 09

1) PCR Biobrick Taz→ run gel→ extract→ ligation into pGEM


 * 20μL H2O
 * 25 μL mastermix
 * 1 μL template Taz miniprep 5 from 8/11
 * 2 μL forward BB primers
 * 2 μL reverse BB primers

PCR program: (i) 94°C 5 mins (ii) 94 °C 30 sec (iii) 53.5 °C 30 sec (iv) 72 °C 90 sec (v) Repeat 2-5 34x (vi) 72 °C 5 mins (vii) Hold 4 °C


 * Nanodrop concentrations of gel extractions of Taz BB PCR:
 * Taz BB PCR1: 57.2 ng/μL
 * Taz BB PCR2: 66.7 ng/μL

2) Digest Taz 1 minipreps (pGEM) wih Bam and Eco→ gel extract

(1)	:95.70 (2): 89.64 (3): 164.52 (4): 150.93 (5): 198.37 (6): 76.46 (7): 86.61 (8): 99.79 Digest at 37°C for two hours. 11:40 am-1:40 pm
 * Taz 1 miniprep (8/16 samples): Nanodrop concentrations (ng/μL)

3) Ligate Taz 1 into pGEm


 * Use Team 1’s gel extraction of pGem backbone(17.5 ng/μL), digested with B, E
 * Ligate digested Taz1 into pGem:
 * 3 μL insert, 1 μL vector
 * 1 μL ligation buffer (10x)
 * 1 μL ligase


 * Use optimized gel extraction protocol: load 2 20 μL samples in separate wells
 * Cut as close as possible for each band→ combine in 1 tube
 * QiaQuick Protocol notes: no unnecessary steps

4) Liquid culture at 5 pm of Taz1-pGem ligations 5) Geneart order

August 18, 09

1) Miniprep of Taz-pGEM 2) Run gel of digest of Bam-Eco on Taz-pGEM→gel extract→ ligation into pNoTat


 * Lane 1: 1 kb ladder
 * Lane 2: digest #1
 * Lane 3: #2
 * Lane 4: -
 * Lane 5: #3
 * Lane 6: #4 (mixture wasn’t 20 μL)
 * Lane 7: -
 * Lane 8: #5


 * Lane 1: 1 kb ladder
 * Lane 2: -
 * Lane 3: #6
 * Lane 4: #7
 * Lane 5: #8
 * Lane 6: -
 * Lane 7: 1 kb ladder
 * Lane 8: -


 * Refer to Taz Bam Eco Digest 8/18.tif for images
 * Digests 1,2,3,6,7,8, came out nicely


 * Gel extraction:
 * Mass (g), Mass+ gel (g), Gel (g), QG buffer:
 * (A) Tube 1-2: 1.0201, 1.1442, 0.1241, 372.3
 * (B) Tube 3: 1.0106, 1.0767, 0.0661, 198.3
 * (C) Tube 6-8: 1.0213, 1.2399, 0.2186, 655.8


 * Nanodrop concentrations: ng/μL
 * A: 8.9
 * B: 8.7
 * C: 15.9

3) Transformation of Taz BB pGEM 4) Stratagene

August 19, 09


 * Ligation of TazBB-pGEM (10:30 am -10:45 pm)

1) 6.19 μL H2O 2) 1 μL 10x buffer 3) 1 μL pGEM 4) 1.31 μL Taz 5) 0.5 μL DNA ligase


 * Transformations into RU 1012

1) Taz pNoTat (4)+C 2) TazBBpGEM (4)+C

August 20, 09


 * Liquid culture: Testing RU1012 (Kanr) with Taz 1 (Ampr)
 * Negative: no growth
 * Amp: no growth
 * Amp+Kan: growth
 * Kan: growth
 * Chloremphenicol: no growth
 * A+K+C: no growth

Week 10

1. 8/24/09: Received successful sequencing for Taz1 (p-GEM). 2. Received GINKGO ompC-RFP (Tet):


 * Incubated plates, inoculated cultures, mini-prepped DNA

3. Constructed Taz1 BB:
 * Digest (EcoR1, Pst1) and gel extraction of mini-prepped Taz1 BB (p-GEM)


 * Taz1 BB (p-GEM) Mini-Preps (Nanodrop Concentrations): Sample: Concentration, 260/280
 * 1: 286.0, 1.93
 * 2: 127.3, 1.98
 * 3: 330.1, 1.92
 * 4: 292.3, 1.95
 * 5:	99.6, 1.98
 * 6: 415.1, 1.92
 * 7: 110.5, 1.94

Gel: Taz1 BB (p-GEM):


 * Gel Extraction Concentration: Taz1 BB (p-GEM): Sample: ng/uL, 260/280
 * 1: 9.8, 1.48
 * 2: 13.7, 1.78

Gel: BB Vector:
 * Digest (EcoR1, Pst1) and gel extraction of BB vector.


 * Gel Extraction Concentration: BB Vector
 * 18.1 ng/uL, 260/280 = 1.83


 * Overnight ligation of Taz1 BB and BB vector.


 * For 50 ng BB vector (2.78 uL of 18.1 ng/uL BB vector Gel Extract) (1458/2079) (3 or 6) = 105.19ng (10.73 uL of 9.8ng/uL Taz1 BB (p-GEM) Sample 1 Gel Extract) or 210.39 ng (21.47uL) Taz1 BB.


 * Transformation of ligation into DH5α. Incubated plates, inoculated liquid cultures, mini-prepped DNA, received successful sequencing.

4. Stratagene Mutagenesis:

(i) Thaw dNTP mix once; prepare single-use aliquots (-20°C) (ii) Control Reaction


 * 5 μL 10 x rxn buffer; 2 μL pwhitescript control plasmid (4.5 kb); 1.25 μL control primers (1), (2); 1 μL dNTP mix; 38.5 μL ddH2O
 * After: 1 μL pFuUltra DNA polymerase


 * Reaction:
 * 5 μL 20x buffer; DNA template (Taz pNoTat); primer (1 mut), primer (2 mut); 1 μL dNTP; X μL ddH2O
 * After: 1 μL PfuUltra DNA polymerase


 * Transformation→ plate: no colonies for both control snad samples

5. Tested Sequencing of Taz1 (p-NOTat ):


 * Digest (BamH1, EcoR1) and received successful sequencing.


 * Taz1 (p-NOTat) Mini-Preps (Nanodrop Concentrations): Sample: Concentration, 260/280
 * 1: 178.4, 1.97
 * 2: 188.6, 1.96
 * 3: 168.7, 1.96
 * 4: 371.2, 1.91
 * 5: 197.2, 1.95
 * 6:	137.5, 2.00
 * 7: 198.4, 1.97

Week 12

September 10, 09

(i) control:
 * 5 μL 10x reaction buffer
 * 2 μL pwhitescript
 * 1.25 μL control primer 1
 * 1.25 μL control primer 2
 * 1 μL dNTP
 * 38.5 μL ddH2O
 * then 1 μL PfuUltra HF DNA polymerase

(ii) sample (same except 2 μL pNoTat template 6 (137.5 ng/μL)


 * Result: no colonies

September 13, 09


 * Transformation of RU1012
 * T1: RU1012+ OmpC-RFP→ Tet plate (3:20 pm)
 * T2: RU1012+ OmpC-RFP→ Tet plate (3:20 pm)
 * T3: RU1012+ OmpC-RFP + Taz→ Tet-Amp plate (3:50 pm)
 * T4: RU1012+ OmpC-RFP+ Taz→ Tet=Amp Plate (3:50 pm)
 * T5: RU1012→ Tet plate (3:20 pm)

Week 13

September 14, 09, 7 am


 * Check plates: RU1012 negative control→ lots of growth!!! Batch of plates is bad.
 * (200 μL) RU1012+OmpC-RFP→ lots of growth
 * (50 μL) RU1012+ OmpC-RFP→ lots of growth
 * MC IPTG+ Xgal→ no growth
 * Sample 1 250 μL → no growth
 * Sample 2 250 μL→ 3 colonies
 * TC IPTG+ X-gal→ 6-7 colonies
 * RU1012+Taz+Ompc-RFP 1→ no growth
 * RU1012+ Taz+ OmpC-RFP 2→ growth

September 15 -16, 09


 * Testing Aspartate binding of Double transformants
 * 8:15 pm: start 10 liquid cultures in minimal media with Amp, Tet, Kan
 * 10:15 am: move 0.5 mL to new 5 mL culture tube (minimal media+ A, T,K)
 * Liquid cultures didn’t grow
 * Redid cultures→ 16 of them

(i) LB→ growth (ii) LB+A→ growth (iii) LB+T→growth (iv) LB+A+T→ no growth (v) MM→ no growth (vi) MM+A→ no growth (vii)	MM+T→ no growth (viii)	MM+A+T→ no growth


 * Plates (LB amp, tet-new plates)
 * IPTG induced: re-streak


 * IPTG induced RU1012 Double transformation
 * OmpC-RFP (Tetr)
 * Taz pNotat miniprep1 from 8/21/09(Ampr)


 * Redid double transformation of RU1012 with Taz-pNoTat+OmpC-RFP
 * 2:30 pm: visualized under fluroscope→ WE SEE RED!! DOUBLE TRANSFORMATION SUCCESS.


 * Liquid cultures (picked only red colonies) 5 μL
 * LB (negative)→ growth
 * LB→ growth
 * LB+A→ growth
 * LB+T→ no growth
 * LB+A+T→ no growth
 * MM (neg)→ no growth
 * MM→ no growth
 * MM+A→ no growth
 * MM+T→ no growth
 * MM+A+T→ no growth

September 18, 09


 * To do: try liquid cultures with less Tet
 * Single transformation of RFP (TetR) Construct→RU1012+ DH5α
 * Plate on only Tet plate (make sure to use the TetR construct is usd and not the first KanR construct we received)

Week 14

September 21, 09
 * Retry liquid cultures from LB-A-T plate
 * Amp	Tet	Growth
 * -	-	Yes
 * -	0.1 μL  Yes
 * -	0.5 μL  Yes
 * -	1 μL	  Yes
 * - 	2 μL  Yes
 * - 	4 μL  Yes
 * 5 μL  0.1 μL   Yes
 * 5 μL  0.5 μL   Yes
 * 5 μL  1 μL   Yes
 * 5 μL  2 μL   Yes
 * 5 μL  4 μL   Yes


 * IPTG induction (5 μL) at 10:25 am
 * 0.5 mL liquid culture
 * 5 mL Minimal media
 * 5 μL IPTG


 * Result: All fluoresced red…there is lactose in LB.

September 23, 09


 * Minimal media liquid cultures (1pm→ 10 am)
 * MM (neg)→ no growth
 * MM (pos)+ RU1012 with Taz 1 from 8/20→ growth
 * MM with double transformants→ growth
 * MM+A with double transformants→ no growth
 * MM+T with double transformants→ no growth
 * MM+A+T with double transformants→ no growth

1) no IPTG no aspartate 2) no IPTG 100 μL 0.1M Asp (2mM) 3) 5 μL IPTG 50 μL Asp (1mM) 4) 5 μL IPTG 100 μL Asp (2mM) 5) 5 μL IPTG 10 μL Asp (0.2 mM) 6) 5 μL IPTG (3 hours later)→ 10 μL Asp 7) 5 μL IPTG (3 hours later)→ 50 μL Asp 8) 5 μL IPTG (3 hours later)→ 100 μL Asp
 * Testing for RFP (5mL cultures)


 * 0.1 M Aspartate solution: 1.33 g in 100mL dH2O