Editing 1152 pET-Exo-Taq-sso7d (section)

=== Cloning  ===

Date:&nbsp;June 13, 2012 <br> 

Ran two PCR&nbsp;reactions (a)&nbsp;and (b), ran gel in lanes 5-6 and 7-8, respectively. <br> 

(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)<br> 

(b)&nbsp;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)<br> 

[[Image:0613 Amplifacation.JPG]]<br> 

Cut out 1152(a)&nbsp;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. <br> 

[[Image:0613 Reamp Digestion.JPG]]&nbsp; <br> 

Date:&nbsp;6/14/2012<br> 

Redid amplifaction, see gel lanes 4 and 5<br> 

[[Image:0614 Amp.JPG]]<br> 

(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. <br> 

[[Image:0614 amp 2.JPG]] 

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

Date:&nbsp;June 15, 2012 

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

[[Image:1152 Amp Gibson.JPG]]<br> 

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

[[Image:1152 0615.JPG]]<br> 

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

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

UPDATE:&nbsp;Restart from the beginning, PCR&nbsp;using Takala enzyme 

Date: June 18, 2012 

[[Image:1152 Amp 0618.JPG]] 

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

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

[[Image:0618 Gibson.JPG]] 

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

Cut out bands for DNA&nbsp;purifaction, digest with Nde1 + Kpn1, clone into Nde1 + Kpn1 digested 368 pET30b-sso7d-KX<br>

[[Image:0618_Digest.JPG]]<br>

{| width="200" cellspacing="1" cellpadding="1" border="1"
|-
| Lane<br>
| Sample<br>
| Enzyme<br>
|-
| 1<br>
| Gibson Amp 1152<br>
| Nde1 + Kpn1<br>
|-
| 2<br>
| pET30b-sso7d-KX<br>
| Nde1 + Kpn1<br>
|-
| 3<br>
| PCR&nbsp;Amp 1152<br>
| Nde1 + Kpn1<br>
|-
| 4<br>
| pET30b-sso7d-KX<br>
| Nde1 + Kpn1<br>
|}

<br> <br>