Template:Pitt/labnotebook3

Restriction Digest of PCR Product with XbaI and PstI

Thursday, July 3, 2014

Result

1. 8.6 ug/mL for RBS PCR product

2. 11.5 ug/mL for no RBS PCR product

Mini-Prep

Tuesday, July 8, 2014

Mini-prepped 5x5 mL of E. coli dam- pBRES36a in LB+amp

Ran out of lysis solution ,unable to mini prep the remaining 25 mL

Yield:

5x50 mL = 250 mL

Concentration: 42 ug/mL, equivalent to10.5 ng plasmid

Testing old Tubes and Preparing LB Agar (100mL)

Wednesday, July 9, 2014

Testing Old Mini-prep Columns and Tubes:

Use 20 uL of 3.0 ug/mL DNA

Result

New tubes: 52 ug/mL

Old Tubes: 20 ug/mL

Conclusion: old tubes can no longer be used purchased new tubes

100 mL LB Agar and autoclaved:

1g tryptone

0.5g yeast extract

1g NaCl

1.5 g Agar (1.5%)

100 mL Water (ddH2O)

50 mL aliquots

Ligating Hsp60 Inserts Into Vector Backbone

Monday, July 14, 2014

1. Digestion

Materials:

Procedures:

1. Mixed materials together in microcentrifuge tubes

2. Incubate 30 minutes at 37 degrees Celsius

2. PCR Purification (using Promega Wizard SV gel clean up kit)

3. Ligations

a. Ligation 1: RBS and Vector

b. Ligation 2: no RBS and Vector

Materials for Ligation 1:

Materials for Ligation 2

Procedures:

1. 15 minutes at room temperature

2. Put on ice until transformation

4. Transformation (see transformation protocol)

Housekeeping

Monday, July 14, 2014

30 mL LB Agar and autoclaved:

0.3 g tryptone

0.18 g yeast extract

0.3 g NaCl

0.45 g Agar (1.5%)

30 mL Water (ddH2O)

Chloramphenicol (CAM)

10 mg in 1 mL of 95% EtOH

5ug/mL workign concentration is needed

Used 35 ug/mL final

Prep 50 mL LB agar +CAM:

0.5 g tryptone

0.25 g yeast extract

0.5 g NaCl

0.75 g Agar (1.5%)

48 mL Water (ddH2O)

25 uL of 10 mg/mL CAM

Competent cells growing

Housekeeping and Ligation

Wednesday, July 16, 2014

Prep 75 m

0.75 g tryptone

0.375 g yeast extract

0.5 g NaCl

1.125 g Agar (1.5%)

50 mL Water (ddH2O)

Ligations

Ligation 1: RBS and Vector

Ligation 2: no RBS and Vector

Materials for Ligation 1:

Materials for Ligation 2

Transformation:

50 mL comp cells + 5 uL plasmid (shipment plasmid + RBS/no RBS)

On ice for 2 minutes

Heat shock 42 degree Celsius for 45 seconds

Ice 2 minutes

550 mL SOC media into mixture

Shake at 37 degree Celsius for 1 hour (recovery)

Plate onto CAM plate + incubate at 4 pm

Plan + CAM plate

Friday, July 18, 2014

Plan of Attack with pBRES36a

1. Digest with individual restriction enzymes and a negative control

a. No enzyme

b. XbaI

c. PstI

d. SpeI

e. EcoRI

2. Visualize on Gel

3. Double Digest with Pair from Step 1

4. Visualize on gel

5. Map the sequence

CAM plates made with 35 ug/mL

Transformation with:

1. Ligation product (2 of them)

2. Plasmid that's known to express CAM (for control)

7/20/14

2^nd Double Digestion: For the purpose of confirming that there was no mix up in the previous digestion.

Housekeeping

Wednesday, July 23, 2014

Cam plates: 35 ug/mL, dilute 1:1000

Used 75 mL, used 75 ul

For 4 plates

Transformation with RBS:

50 mL E. coli comp cells, 5 uL RBS ligation

50 mL E. coli comp cells, 5 uL no RBS ligation

Transformed and recovered for 1 hour

Plated 50 uL of transformed cells onto cam plates

Restriction Digest of pBRES36a Plasmid

Monday, July 28, 2014

1. Digestion

Purpose: cut with restriction enzymes to provide a rough map for the pBRES36a plasmid and confirm the plasmid's identity

Materials:

Procedures:

1. Mix reagents together and spin down gently.

2. Incubated for 10 minutes

3. Pipet up and down gently to mix samples

4. Load each sample onto the agarose gel

5. Run electrophoresis according to the conditions specified.

Enzyme Used:

1. XbaI

2. PstI

3. SpeI

4. EcoRI

2. Gel Electrophoresis

Run 1: Gel Set Up (7/18/14)

Run 1 Condition:

1. 2.0 hours

2. 80 Volts

3. 0.8% Agarose Gel

4. 1x TBE Buffer

Run 2: Gel Set Up (7/28/14)

Run 2 Condition:

1. 1.5 hours

2. 80 Volts

3. 0.8% Agarose Gel

4. 1x TBE Buffer

July 28, 2014 con’t:

Results

Run 1 (7/18/14) Left and Run 2 (7/28/14) Right

Ligation, Transformation, Selection

Thursday, July 24, 2014

Ligations

Ligation 1: RBS and Vector

Ligation 2: no RBS and Vector

Materials for Ligation 1:

Materials for Ligation 2

Incubate at RT for 15 minutes

Transformation

1. 50 mL E. coli comp cells

2. 6 uL plasmid

3. 2 min on ice

4. 45s heat shock at 42 degrees Celsius

5. 2 min on ice

6. 950 mL SOC growth media

7. 1 hour recovery at 37 degrees Celsius

Selection:

plate 60 mL of transformed cells onto selection plate (LB + CAM plates)

8/1/14

1. Take out overnight liquid culture around 11:00 am

2. Mini prep iGEM plasmid parts

8/4/14 Lab

Make 1% Gel (30 mL):

0.3 g Agarose

30 mL 1x TBE

1 uL EtBr

Restriction Digests:

hsp60 PCR products:

RBS: no RBS:

6 uL ddH₂O 6 uL ddH₂O

2 uL Buffer 2 uL Buffer

1 uL XbaI 1 uL XbaI

1 uL PstI 1 uL PstI

10 uL PCR product 10 uL PCR product

iGEM Parts:

mCherry: Cathelicidin: deSaturase:

6 uL ddH₂O 6 uL ddH₂O 6 uL ddH₂O

2 uL FD Green Buffer 2 uL FD Green Buffer 2 uL FD Green Buffer

1 uL XbaI 1 uL XbaI 1 uL XbaI

1 uL PstI 1 uL PstI 1 uL PstI

10 uL mCherry 10 uL Cathelicidin 10 uL deSaturase

PCR Purification:

Run PCR purification of hsp60 digests (RBS and no RBS)

1: RBS concentration-

2: no RBS concentration –

Gel Electrophoresis:

Ladder – mcherry – cath – desat - empty - cath – mcherry – desat

Failed. Will upload pic later

Ligation:

Ligate hsp60 RBS and no RBS into pSB1C3 backbones

RBS: no RBS:

9.5 uL ddH₂O 10.5 uL ddH₂O

3.5 uL backbone 3.5 uL backbone

4 uL insert 3 uL insert

2 uL buffer 2 uL buffer

1 uL ligase 1 uL ligase

Transformation:

Transform RBS and no RBS ligations into E. coli competent cells

5 uL plasmid

100 uL competent cells

Ice 2 minutes

Heat shock (42C) 45s

Ice 2 minutes

Add 950 uL outgrowth media

Recover with shaking (37C) for 1 hour

Plate onto selection plate (chloramphenicol)

Restriction Digest:

Cut mCherry prep, Cath. Prep., deSat. Prep, 1 RBS, 1 no RBS, 1 mCherry with XbaI and PstI

6 uL ddH₂O

2 uL FD Green Buffer

1 uL XbaI

1 uL PstI

10 uL plasmid DNA

Gel Electrophoresis:

1 kb+ ladder – mCherry prep – Cath. Prep – deSat prep – 1 RBS – 1 no RBS – 1 mCherry -

8/5/15 Lab

Make 1% Gel (35 mL)

0.35 g Agarose

35 mL 1x TBE

1 uL EtBR

Restriction Digests:

Master Mix (for 6 reactions): x6.5 =

6 uL ddH₂O 39 uL

2 uL Green Buffer 13 uL

1 uL XbaI 6.5 uL

1 uL PstI 6.5 uL

Combine 10 uL of Master Mix with 10 uL of each plasmid:

mCherry Prep, Cath. Prep, deSat prep., 1 RBS, 1 no RBS, 1 mCherry

Incubate 45 mins at 37C

Gel Electrophoresis:

Run 1 hour 30 mins

Lanes:

1 kb+ ladder – mCherry mini – Desat – Cath – 1 RBS – 1 no RBS – 1 mCherry – 1 kb+ ladder

08/08/14

Used the culture collected from 8/1/14 mini-prep for the subsequent restriction digest

1. Restriction digest (45 minutes)

2. Run agarose gel

Digestion was incomplete and need to repeat experiment

Experiment was repeated on 8/9/14 and digestion was successful. The desire bands were excised and purified. Unfortunately the image file was lost due to a technical problem prior to routine file backup. However, further experiments continued to use parts isolated on this date and bands of correct size were present through all of these steps.

Lab Work 8/12/14:

Gel Purification:

*Add 10 uL of Membrane Binding Solution per 10 mg of Gel slice

-Vortex Gel and solution

-Incubate at 55C for 10 mins to melt gel

-Spin Down

-Gel Purify

Miniprep:

Miniprepped mRFP1 and MelA liquid cultures

-used same elution buffer for two of the same sample to obtain double DNA concentration

Concentrations:

1 mRFP: 516 ng/uL

2 mRFP: 462.5 ng/uL

3 mRFP: 243.5 ng/uL

1 MelA: 117.9 ng/uL

2 MelA: 158.3 ng/uL

3 MelA: 146.1 ng/uL

Lab Work 8/13/14:

Restriction Digest:

Digesting 1 mRFP, 2 mRFP, 3 mRFP, 1 MelA, 2 MelA, 3 MelA with XbaI and PstI.

20 uL Reactions: Master Mix:

5 uL Plasmid DNA 13 uL FD Green Buffer

2 uL FD Green Buffer 6.5 uL XbaI

1 uL XbaI 6.5 uL PstI

1 uL PstI 78 uL ddH₂O

12 uL ddH₂O 104 uL Total

*Reactions are 5 uL Plasmid + 15 uL Master Mix

*There are 6 reactions. Master Mix is 6.5x

*Incubate for 45 minutes at 37C

Gel Electrophoresis:

Running a gel to check the validity of the mRFP1 and MelA mini-prepped DNA.

Expect to see mRFP1 band at 706 bp and MelA band at 1844 bp.

Lanes:

1 kb+ --- 1 mRFP --- 2 mRFP --- 3 mRFP --- 1 MelA --- 2 MelA --- 3 MelA --- EMPTY

Gel: