User:Neil J. Parikh/Lab Notebook

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Pipetting & Making Solutions

Pg. 6

              I.        Pipetting

a.    Learned to use pipettes properly

b.    Make sure to wipe down pipettes between solutions (with Ethanol)

c.    Change tips every time you go between solutions

            II.        Solutions

a.    Task: Make 0.3M KCl

                                                  i.    Salt (KCl): CAS 7447-40-7

                                                ii.    MW: 74.56g/mol

                                               iii.    2.2368 g / 100mL

 

Plate Creation & Cell Culture

Pg. 7

              I.        Steps for creating a plate:

a.    Dissolve 25g LB in 800mL of H2O

Bring the final volume to 1L in a 2L flask

Add 15g Bactoagar, cover with foil, autoclave for 20-30 minutes

b.    Bring back to lab, cool to 50°C while stirring

c.    Add100mg AMP & let swirl until dissolved

d.    Pour it into the plates (about 2-3mm deep)

            II.        Cell Culture:

a.    Use solution of LB with AMP

Ensure to keep the bags sealed to keep everything sterile

*Note once a bag of plates has been opened, the whole thing must be used

The tubes allow the solution to be aerated

The tubes with bacteria should look cloudy upon visual inspection—can be confirmed with cell density measurement (using the Spec)

b.    Sterilize the autopipette, set to 1mL (make a 2mL culture, however)

Add 2mL of the LB (for a miniprep); you will have to add 1mL, two times

c.    Label one as the control and another as your experimental sample

*Label the tubes with your Initials, Sample Name (control or experimental) and the date

d.    Dip the culture scraper in ethanol and flame lightly (2x) to sterilize

e.    Let the scraper cool for a few seconds

f.     Scrape off a tiny bit of bacteria off of the plate and place the scraper into the tube and shake around to ensure the bacteria breaks off into the tube

g.    Set incubator at 37°C overnight and let shake gently for 12-14hrs

 

Transformation of E.coli with Recombinant DNA

Pg. 9

*note, the heat shock to the tubes is very important for transcription to occur

+ Controls:

BBa_I13522 : pTetGFP

BBa_J45220

Group 1:

BBa_R0011

BBa_J45219

Group 2:

BBa_I14032 / BBa_R0051 (same)

BBa_J06702

Group 3:

BBa_J04500

BBa_J04630 / BBa_I13401

 

Rima & I did the transformation for Group 2:

                              I.        BBa_I14032

a.    Name: promoter P(Lac) IQ

b.    Sequence: promoter (-35, -10 bases before coding)

c.    Location: 2007, Plate 2, Well 17K

d.    Plasmid: pSB2K3

                            II.        BBa_R0051

a.    Name: promoter (lamda cl regulated)

b.    Sequence: R0051 promoter

c.    Location: 2007, Plate 1, Well 9C

d.    Plasmid: pSB1A2

                           III.        BBa_J06702

a.    Name: mCherry, bacterial with RBS & forward terminator

b.    Sequence:

RBS

mCherry

Terminator

Terminator

B0034

J06504

B0010

B0012

c.    Location: 2007, Plate 2, Well 11D

d.    Plasmid: pSB1A2

                          IV.        Cells Used: E.coli: JM109 (Promega); Catalog #: L2001

 

                           V.        3 Plates Made:

a.    Control: 100μL LB / Cell

b.    Test Plate 1: 100μL LB/ Cell

c.    Test Plate 2: 1μL cell : 99μ H2O

 

Actual Transformation Procedure:

                              I.        If using DNA from the registry, dilute with 9μL of H2O. If using our own DNA, use 200ng.

a.                inject the water directly into the well, titerate, and then pipette it out

                            II.        Thaw 100μL competent cells on ice. (50 μL with our own/ 100 μ if commercial cells used)

                           III.        Mix competent cells with 50 μ CaCl2 (only if using our own cells)

                          IV.        Add 4 μL of registry DNA, or 200ng of our own DNA to the cells.

                           V.        Put in the ice bath for about 20 minutes.

                          VI.        Remove and immediately put in the hot water bath (42°C) to heat shock for 45-60 sec.

                         VII.        Place back in the ice bath for 2 minutes.

                       VIII.        Add 900 μL ice cold, antibiotic free LB to the cells

                          IX.        Incubate at 37°C for 90 minutes in the shaking incubator.

                           X.        Dilute as desired, make the necessary controls, and plate cells using 100 μL / plate. (200 if not commercial)

                          XI.        Incubate upside down (so that the label you wrote on it is face up) at 37°C for 18-24 hours, then refrigerate.

a.                The controls should not have bacteria growing on them!

 

Miniprep

Pg. 12

*Start with Parts: E0241 (x3), R0010, R0040, C0040 from the registry*

®    E0241: PoPs to GFP Converter

§  RBS: B0032

§  GFP: E0040

§  Terminator: B0016

®    R0010: Promoter Lac I (Regulator)

®    R0040: Promoter (tetR, negative)

®    C0040: Tetracycline repressor from transposon Tn10 (+LVA) [note that +LVA is a degradation tag]

 

Look out for:

Control should not be cloudy at all, culture should have gas bubbles, should be cloudy, especially at the bottom

*If you start seeing sediment at the bottom, the cells may have lysed and let proteins into the tube*

Procedure (General):

Use the QIAGEN Kit to separate the plasmid DNA (so it should not have much chromosomal DNA)

                              I.        Grow cells for 12 to 14 hours

                            II.        Suspend cells in

                                                  i.    buffer 1 (with glucose & EDTA)

                                                ii.    buffer 2 (SDS-detergent, NaOH) [SDS gets rid of the membrane and NaOH denatures the plasmid and DNA]

                                               iii.    buffer 3 (K Acetate / Acetic Acid)

-things become an insoluble precipitate

-renature the DNA/plasmid

-only the plasmid DNA will renature properly

-the plasmid DNA remains soluble & the chromosomal DNA will become the precipitate

                           III.        Columns: use EtOH to extract DNA

 

Procedure (Specific):

*make sure to check your buffers for salt precipitation—this may indicate a change in concentration*

                              I.        Get solution and put 1.5mL into an Eppendorf tube to centrifuge

-after centrifuging for 30 seconds @ 13k RPM, remove the supernatant

-add more solution, and centrifuge again using same procedure

                            II.        Add 250 μL PI to tube after the supernatant is fully removed

                           III.        Add 250 μL P2, and invert 4 to 6 times

-immediately add 350 μL N3, and invert again 4 to 6 times

                          IV.        Dispose of the flow through, add 0.5mL of buffer PB (that is milliliter not microliter!) then centrifuge for 30-60 seconds

                           V.        Add 750 μL PE, then centrifuge again, remove the waste, centrifuge again

                          VI.        Add 50 μL BE buffer

                         VII.        Measure the DNA concentration using Nanodrop

                       VIII.        Ethanol Precipitation (see separate procedure)

 

Ethanol Precipitation (for >200bp)

Pg. 15

*used to purify the DNA (really small fragments) or to increase the concentration of it

                      I.        Add:

                                          i.    3 volumes of COLD 100% EtOH

                                        ii.    1/10 volume of 3M sodium acetate

                                       iii.    1 μL GlycoBlue

                    II.        Incubate for 1 hr @ 80°C

                   III.        Centrifuge for 30min @ 0°C

                  IV.        Remove supernatant

                   V.        Air Dry

                  VI.        Resuspend in H2O

 

Specific Procedure Example:

For 50 μL:

Add 150 μL EtOH

20 μL NaAc.

For 100 μL:

Add 300 μL EtOH

40 μL NaAc.

 

Gel Electrophoresis

Pg. 16

*applying an electric field over the gel will separate fragments by size

*compare the movement of the DNA to the ladder

*ensure that the well is on the negative side!*

 

®    DNA has a (-) charge because of the phosphate groups

®    moves toward the anode (+) and away from the cathode (-)

®    DNA is separated by size, distance ~ 1 / molecular weight

§  100bp-50kbp

®    Gel acts as a sieve: 0.3% large fragments to 2% small fragments

®    after the gel is made, put it in the microwave for 1minute, then cool the bottle in H­2O, and take the lid off

 

Casting Gel:

1% Agarose, 0.5g Agarose

50mL TBE + SYBR Safe

 

Dilute the 5xTBE to 0.5x

 

Add 2 μL of the loading dye!

 

Ladder Formula:

1 μL 1kbase ladder

2 μL BlueJuice Dye

7 μL H2O

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