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

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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.


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
HA130EcoRI (1)SpeI (1)FD Clr (3)0
EM730XabaI (1)SpeI (1)FD Clr (3)0
v012015EcoRI (1)SpeI (1)FD Grn (3)10
v012015XabaI (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:

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
v0120 E/S digest9.52.01
v0120 X/S digest9.91.76

Vector and insert mass calculations:

Vector Vector size (bp) Vector amt (ng) Insert:Vector Ratio Insert Insert size (bp) Insert amt (ng)
v0120 E/S3244502HA1 E/S1203.70
V0120 X/S3244502EM7:ZeoR X/S45013.87

Vector volume calculations:

Vector Vector amt (ng) Vector conc. (ng/µL) Vector vol. (µL)
v0120 E/S509.55.26
V0120 X/S509.95.05

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)2200
Vector DNA (50 ng)
2x Roche Rapid Ligation buffer8.
New England Biolabs T4 ligase1111

Incubate at room temperature, 10 minutes.

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)

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