1152 pET-Exo-Taq-sso7d
Cloning of pET-Exo-Taq-sso7d
[edit]Purpose: Cloning of pET-Exo-Taq-sso7d
Date: 2012-06-13
Notebook:
Page:
Clone ID: 1152
Experimenters: Jennifer Truong
Results
[edit]Cloning
[edit]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
[edit]
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.