1151 pET30b-sso7d-ToughTaq

Cloning of pET30b-sso7d-ToughTaq

Purpose: Cloning of pET30b-sso7d-ToughTaq by gene synthesis

Date: 2012-06-13

Notebook: 

Page:

Clone ID: 1151

Experimenters: Jennifer Truong

Results

Cloning

Date: June 13, 2012

Synthesized template in 3 fragments (KTaq1, KTaq1 and KTaq3), PCR with KAPA enzyme for 5 cycles with 15 seconds extension time, conbined them, added first (KTaq1-1) and last (KTaq3-14) oligos , PCR with KAPA enzyme for 20 cycles with 45 seconds extension time. Ran gel (lanes 1-4), supposed to be ~1.7 kb

Reamplified 1151 (see lanes 1-3) in fragments

Lane 1: KTaq1-1 - KTaq1-16 (636 bp)

Lane 2: KTaq2-1 - KTaq2-16 (629 bp)

Lane 3: KTaq3-1 - KTaq3-14 (487 bp)

 

Unsuccessful

Date: June 14, 2012

Reamplified PCR product, see gel lanes 1-3

Lane 1: KTaq1-1 - KTaq1-16 (636 bp)

Lane 2: KTaq2-1 - KTaq2-16 (629 bp)

Lane 3: KTaq3-1 - KTaq3-14 (487 bp)

Successful amplifacation, cut out each band, purify each, join them together by Gisbon (~1.67 uL of each fragment, ran gel of joined template

Expected size (~1.9 kb)

Subcloing ToughTaq from 1094 pET30b-ToughTaq to pET30b-sso7d-NK 369 using Kpn1 + Xho1

Digested ToughTaq and vector 369 with Kpn + Xho1, cut out bands, and purified DNA.

Ligating over night

Date: June 15, 2012

Transformed the ligated DNA from yesterday with DH5 alpha cells and plated on KAN plates

Also, redid the PCR amplication using PHUSION to join the fragments (1 min 30 sec extension time, 20 cycles) instead of Gibson

Cut out band (~1.9 kb) and purified DNA, Digested ToughTaq and vector 369 with Kpn + Xho1

Cut out the digestion (see lanes 3 and 4), purified and ligated for 2 hours
Transformed the ligated DNA with DH5 alpha cells and plated on KAN plates

Date: June 22, 2012

Combined by Gibson, no band (expected: 1.9kb)

Date: June 25, 2012

Ran gel for PCR product of fragments joined with PCR and ligase

No clear band where it is supposed to be (expected = 1.9 kb)

Colony Screening

All negative, need to reclone

Digested 1151 with enzymes, incorrect band size (lanes 5 and 6)

Date: June 21, 2012

Digested 1151 with enzymes, did not run gel long enough. Redigest sample 5 (lane 5), may be the correct size?

Redigested samples 5 and 6, band is supposed to be 1.7 kb, may send sample 5 to sequencing

Cloning of 1151 pET30b-sso7d-ToughTaq

Purpose: Cloning of 1151 pET30b-sso7d-ToughTaq

Date: 2012-7-9---2012-7-12

Notebook:0

Page:

Clone ID:1151

Experimenters: yingying

Results

cloning

Gene synthesis 3 fragments,

lane1: KTaq1-1+KTaq1-16;

lane2: KTaq2-1+KTaq2-16;

lane3: KTaq3-1+KTaq3-14;

lane1:PCR using phusion

tempalte:PCR1+PCR2+PCR3

primer:KTaq1-1+KTaq3-14;

	lane 3	lane 4
	PCR product	pET30b-sso7d-NK 369
enzymes	Kpn1 + Xho1

Colony Screening

Date: 2012-7-12

Make miniprep and digest with  Kpn1 + Xho1

clone 1,2,3,4,5,6,7,8,9 may be right, need to sequence

Result intepretation/Summary

• <br>

Conclusion

<br>

<br>

<br>