# Rima Shah: my lab notebook

(Difference between revisions)
 Revision as of 16:11, 12 March 2008 (view source)← Previous diff Current revision (18:54, 6 June 2008) (view source) Line 159: Line 159: Incubation of tubes- 2 hours Incubation of tubes- 2 hours *Enough time to allow plasmid DNA to be cut by enzymes *Enough time to allow plasmid DNA to be cut by enzymes + + ==Meeting with Dr. Carlos Aizenman== + 6/06/2008 + + He suggested we: + *calibrate our voltmeter with units for our purpose + *use multi-well plates for our apparatus + + He showed us his lab and helped us design an apparatus involving the use of a voltmeter and stainless steel wire to run several experiments in parallel. + + He lent us a 6-well plate and we plan on creating this apparatus in lab tomorrow.

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

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

```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

## Meeting with Dr. Carlos Aizenman

6/06/2008

He suggested we:

• calibrate our voltmeter with units for our purpose
• use multi-well plates for our apparatus

He showed us his lab and helped us design an apparatus involving the use of a voltmeter and stainless steel wire to run several experiments in parallel.

He lent us a 6-well plate and we plan on creating this apparatus in lab tomorrow.