Rima Shah: my lab notebook

Home        Contact        Lab Notebook        Internal        The Team        Lead Sensor        Tri-Stable Switch       

1/30/08

Pipetting

Exercise 1: 2μl blue solution + 5μl clear

Exercise 2: 20μl blue solution + 70μl clear

Creating Solutions

Create 0.3M KCl solution

In 0.3M, 0.3 mol/L

KCl FW= 74.56g

FW KCl * 0.3M= 22.368 g/L

For solution to fit in provided container, make 100 mL solution

(22.368g/L) / 4= 2.2368g/100mL

Plating

Ingredients LB/Amp plates:

25g LB 15g Agar 800 mL water

Preparation:

• Dissolve 25g LB in 800mL water by swirling tube→ make final volume 1L.
• Pour into 2L flask, add 15g Bactoagar (not agarose) and swirl until completely dissolved.
• Cover top with foil, autoclave 20-40 minutes (autoclave tape should turn black)
• Swirl solution (stirrer at 7) in flask to mix molten agar.
• Cool solution to 50 C (should be able to hold bottom 10-20 seconds)
• Add 100mg Ampicillin, swirl until dissolved.
• Lay out 40 plates, unstacked. Pour prepared solution into plates to depth of approx. 3mm. *To remove surface bubbles, briefly flame surface with Bunsen burner.
• Leave plates out at room temp. unstacked. Wait a few hours before stacking and 24hrs before refrigerating.
• Label plates with date and Amp.
• Store plates in plastic sleeve in refrigerator.

To turn LB plates in LB/Amp plates: Mix 200 ul 5mg/ml stock ampicillin. Add drops ampicillin in several locations around plate so that it’s uniformly distributed. Spread solution around plate with sterile glass rod and allow it to absorb into plate.

Cell Culture

Materials: LB-AMP plate Sterile tubes (loose caps allow in O2)

Know culture is successful if culture becomes cloudy after 1 day

Control: LB-AMP Culture: Plasmid PRS305 +LB-AMP + bacterial culture

Procedure:

• Label tubes: control/test, date, initials
• Fill ea. tube with LB
• Sterilize cell loop- dip in ethanol and briefly run through fire
• Don’t’ open cell plate completely, scoop SMALL amount into tube
• Incubate: 37 C overnight—then slow shaker 12-14 hours

Restriction Digest protocol

DNA Digest Procedure:

• Larger part is always insert (reporter protein etc.) and double digest is used to cut it out of plasmid
• Smaller part remains in vector plasmid and double digest creates place for insert

XbaI/PstI DOUBLE DIGEST (used to remove insert from plasmid) In eppendorf tube: 1) 5 ul BSA

• BSA- Bovine Serum Albumin- stabilizes enzymes and prevents enzymes from adhering to reaction vessel

2) 5 ul Buffer H

• Buffer H- Simulates natural environment of enzymes. Each enzyme combination requires unique buffer with different salt concentrations.*

3) 21 ul DNA (200 ng-1ug)

• The DNA plasmid that contains the reporter (or any protein insert)

4) 1.5ul XbaI

• Cuts plasmid at X site (before the protein insert)**

5) 1.5 ul PstI

• Cuts plasmid at P site (after the protein insert)**

_____________________

TOTAL: 34 ul

SpeI/PstI DOUBLE DIGEST (used to make room on vector for insert) In eppendorf tube: 1) 5 ul BSA

2) 5 ul Buffer B

• Buffer B simulates environment for these specific enzymes*

3) 21 ul DNA (200ng-1 ug)

• The DNA plasmid that will serve as the vector

4) 1.5ul SpeI

• Cuts plasmid at S site**

5) 1.5ul PstI

• Cuts plasmid at P site**

_____________________

TOTAL: 34 ul

1. Buffers for specific restriction enzyme combinations (Manufacturer: Promega)

Eco RI/Spe I: Multicore Eco RI/Xba I: Multicore Buffer H Eco RI/Pst I: Buffer H Spe I/Xba I: Multicore, Buffer E Spe I/ Pst I: NONE (~Buffer B) Xba I/ Pst I: Buffer H

- Each manufacturer has different naming schemes and combinations→ all parts and buffers should be from same company

1. 1. Restriction Site Cut Sequences

(Xba I and Spe I are compatible)

Xba I: T CTAGA AGAGC T

Spe I: A CTAGT TGATC A

Eco RI: G AATTC CTTAA G

Pst I: CTGCA G G ACGTC

Incubation of tubes- 2 hours

• Enough time to allow plasmid DNA to be cut by enzymes