User:Mara Peterson/Notebook/Mara Peterson/2015/12/14

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Overview

Performing restriction digest on parts HA1 and EM7:ZeoR, as well as restriction digest on v0120 plasmid, followed by gel extraction. Then performing a ligation reaction followed by transformation of competent E. coli cells.

Materials/Methods

Homology Arm 1: amplified using P040 and P137, contains EcoRI and SpeI restriction sites. Concentration: 6 ng/µL
EM7:ZeoR: amplified using P136 and P139, contains XabaI and SpeI restriction sites. Concentration: 38 ng/µL
v0120: basic BioBricks-compatible cloning vector. Contains standard restriction sites. Concentration: 100 ng/µL

Following the Assembly 101 procedure, for the most part.


Restriction Digest

Part Volume (µL) Enzyme 1 Enzyme 2 Buffer H2O
HA1 30 EcoRI (1) SpeI (1) FD Clr (3) 0
EM7 30 XabaI (1) SpeI (1) FD Clr (3) 0
v0120 15 EcoRI (1) SpeI (1) FD Grn (3) 10
v0120 15 XabaI (1) SpeI (1) FD Grn (3) 10

Digested at 37°C for 10 minutes, followed by heat inactivation at 80°C for 5 minutes.


Gel Purification
Ran the v0120 parts on a gel at 100 V for 60 minutes. Gel image:

File:2015-12-14 restriction digest and PCR product gels.tif

Cut out the two large v0120 bands (backbone) and performed gel purification using the Sigma kit. Measured concentration on the nanodrop system...

Sample ID ng/uL 260/280
pSB1A3_BsmBI 31.4 1.90
v0120 E/S digest 9.5 2.01
v0120 X/S digest 9.9 1.76

Ligation
Vector and insert mass calculations:

Vector Vector size (bp) Vector amt (ng) Insert:Vector Ratio Insert Insert size (bp) Insert amt (ng)
v0120 E/S 3244 50 2 HA1 E/S 120 3.70
V0120 X/S 3244 50 2 EM7:ZeoR X/S 450 13.87

Vector volume calculations:

Vector Vector amt (ng) Vector conc. (ng/µL) Vector vol. (µL)
v0120 E/S 50 9.5 5.26
V0120 X/S 50 9.9 5.05

Insert volume calculations: Insert Insert amt (ng) Insert conc. (ng/µL) Insert vol. (µL) HA1 E/S 3.7 6.1 0.61 EM7:ZeoR X/S 13.87 37.8 0.37

I'm going to err on the side of caution with the insert volumes and aliquot 2 µL for each one instead of the 0.3-0.6 µL recommended.

Ligation reaction:

Ligation HA1 EM7:ZeoR Neg ctrl (E/S) neg ctrl (X/S)
Insert DNA (X ng) 2 2 0 0
Vector DNA (50 ng) 5.26 5.05 5.26 5.05
2x Roche Rapid Ligation buffer 8.26 8.05 6.26 6.05
New England Biolabs T4 ligase 1 1 1 1
dH2O 0 0 0 0
Total 16.52 16.1 12.52 12.1

Incubate at room temperature, 10 minutes.


Transformation
Follow the traditional transformation protocol.

4 plates:

  • HA1_v0120
  • EM7:ZeoR_v0120
  • HA1 neg control (v0120 E/S digested, no insert)
  • EM7:ZeoR neg control (v0120 X/S digested, no insert)