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!

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