User:JeffreyLau

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(2006/06/15)
(2006/06/15)
Line 5: Line 5:
'''Ligation'''
'''Ligation'''
*Since our e-gel failed, we ligated DNA extract from David and Peng.
*Since our e-gel failed, we ligated DNA extract from David and Peng.
-
**Mixed 6 µL insert (E0241), 2 µL vector (R0010), 2 µL DNA dilution buffer in reaction vial
+
*Mixed 6 µL insert (E0241), 2 µL vector (R0010), 2 µL DNA dilution buffer in reaction vial
-
**Added 10 µL ligation buffer
+
*Added 10 µL ligation buffer
-
**Added 1 µL ligase, mixed
+
*Added 1 µL ligase, mixed
-
**Let incubate 5min at room temperature
+
*Let incubate 5min at room temperature
-
*Transformation
+
 
-
**Mixed 20 µL of ligation product with 30 µL competent cells
+
'''Transformation'''
-
**Negative control: 2 µL of dH20 with 30 µL competent cells
+
*Mixed 20 µL of ligation product with 30 µL competent cells
-
**Positive control: 5 µL of positive control with 30 µL competent cells
+
*Negative control: 2 µL of dH20 with 30 µL competent cells
-
**Incubate on ice for 20min
+
*Positive control: 5 µL of positive control with 30 µL competent cells
-
**Heat shock @42C for 30s
+
*Incubate on ice for 20min
-
**Rest on ice for 2min
+
*Heat shock @42C for 30s
-
**Incubate overnight @37C
+
*Rest on ice for 2min
 +
*Incubate overnight @37C
== 2006/06/14 ==
== 2006/06/14 ==

Revision as of 22:23, 15 June 2006

About

Undergraduate concentrating in Computer Science (class of 2007), member of 2006 Harvard iGem team.

2006/06/15

Ligation

  • Since our e-gel failed, we ligated DNA extract from David and Peng.
  • Mixed 6 µL insert (E0241), 2 µL vector (R0010), 2 µL DNA dilution buffer in reaction vial
  • Added 10 µL ligation buffer
  • Added 1 µL ligase, mixed
  • Let incubate 5min at room temperature

Transformation

  • Mixed 20 µL of ligation product with 30 µL competent cells
  • Negative control: 2 µL of dH20 with 30 µL competent cells
  • Positive control: 5 µL of positive control with 30 µL competent cells
  • Incubate on ice for 20min
  • Heat shock @42C for 30s
  • Rest on ice for 2min
  • Incubate overnight @37C

2006/06/14

Biobrick tutorial

More detailed procedure notes are in lab notebook.

  • Began with 12 samples (3x each of negative control, R0010, E0241, E7104). Fortunately our negative controls showed no growth overnight.
  • Made 9 minipreps for the remaining samples. Set aside the E7104 samples (they are a positive control).
  • Performed digests on 2x each of R0010 and E0241. We lost one of our E0241 samples by accident, so we threw out an R0010 sample to balance.
  • Added Calf Intestinal Phosphatase (CIP) to R0010 samples to prevent self-ligation of the R0010 vector.
  • Performed gel electrophoresis on R0010 and E0241 samples.
    • Our e-gel was not entirely successful.

Image:HH-JL biobrick-tutorial egel.jpg

Must be more careful next time.

Notes on using the nanodrop

  • Nanodrop measures concentration of DNA in small samples (1 µL). It is apparently not very accurate.
  • Clean nanodrop thoroughly with kimwipes and ethanol, before and after each use
  • On desktop, "ND-1000"->"Nucleic Acid"
  • Before each set of samples:
    • Calibrate nanodrop with drop of distilled water
    • Calibrate nanodrop with drop of background (solute), e.g. distilled water or elution buffer
  • Add sample to nanodrop, enter name of sample, "Measure"
    • Note: mix sample before placing on nanodrop
Personal tools