1152 pET-Exo-Taq-sso7d

Cloning of pET-Exo-Taq-sso7d

Purpose: Cloning of pET-Exo-Taq-sso7d

Date: 2012-06-13

Notebook: 

Page:

Clone ID: 1152

Experimenters: Jennifer Truong

Results

Cloning

Date: June 13, 2012

Ran two PCR reactions (a) and (b), ran gel in lanes 5-6 and 7-8, respectively.

(a) PCR fragment using pET30b-DeepVent-ss7d (423) as a template with the primers DeepVent-Exo-Nde1-F and DeepVent-Exo-R (supposed to be ~600 bp)

(b) Synthesized fragment using Wt-Taq-XuYan Tag (462) as template with primers Taq_d289-Nco1-F and Taq_d289-Kpn1-R (supposed to be ~1.7 kb)

Cut out 1152(a) band, reamplified 1152(b), see gel lanes 4 and 5, using Wt-Taq-XuYan Tag (462) as template with primers Taq_d289-Nco1-F and Taq_d289-Kpn1-R (supposed to be ~1.7 kb), cut out bands.

 

Date: 6/14/2012

Redid amplifaction, see gel lanes 4 and 5

(a) PCR fragment using pET30b-DeepVent-ss7d (423) as a template with the primers DeepVent-Exo-Nde1-F and DeepVent-Exo-R (supposed to be ~600 bp)

(b) Synthesized fragment using Wt-Taq-XuYan Tag (462) as template with primers Taq_d289-Nco1-F and Taq_d289-Kpn1-R (supposed to be ~1.7 kb)

Unsuccessful amplification, redid with PHUSION enzyme instead of KAPA.

Cut out fragments in lanes 2 and 3, stored them in box in fridge over night, going to join them by Gibson tomorrow.

Date: June 15, 2012

Cut out bands from lanes 2 and 3, purified the DNA, added 2.5 uL of each purified segment to the Gibson mix (15 uL), incubated at 50 oC for 1 hour

Ran gel for the Gibson product (see lanes 1-2), cut out the faint top band, purified, digest DNA and 368 pET30b-sso7d-KX  with Nde1 + Kpn1

Joined fragments using PCR instead of Gibson (see lanes 1 and 2) , cut out the band, purified, digest DNA and 368 pET30b-sso7d-KX with Nde1 + Kpn1

Gibson digestion (lanes 3 and 4) was unsuccessful because of lost DNA.

UPDATE: Restart from the beginning, PCR using Takala enzyme

Date: June 18, 2012

Lanes one and two were amplified with Takala enzyme, lanes 3 and 4 were amplified using PHUSION enzyme (unsuccessful)

Cut out bands one and two for protein purifaction, join by both PCR and Gibson

Joined lane 1 by Gibson and lane 2 by PCR, expected size = ~2.5 kb

Cut out bands for DNA purifaction, digest with Nde1 + Kpn1, clone into Nde1 + Kpn1 digested 368 pET30b-sso7d-KX

Lane	Sample	Enzyme
1	Gibson Amp 1152	Nde1 + Kpn1
2	pET30b-sso7d-KX	Nde1 + Kpn1
3	PCR Amp 1152	Nde1 + Kpn1
4	pET30b-sso7d-KX	Nde1 + Kpn1

Colony Screening

Grew lanes 1, 2, 4 , 5, 6 and 7 overnight

Digested 1151 with enzymes, did not run gel long enough, redigested samples 1, 2 and 3 (lanes 7, 8 and 9)

See lanes 3, 4 and 5. Bands are not in the correct places. Need to redo.