Jennifer.truong at 19:22, 21 June 2012

2012-06-21T19:22:36Z

New page

== '''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) 

[[Image:0613 Amplifacation.JPG]] 

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. 

[[Image:0613 Reamp Digestion.JPG]]  

Date: 6/14/2012 

Redid amplifaction, see gel lanes 4 and 5 

[[Image:0614 Amp.JPG]] 

(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. 

[[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: 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

[[Image:1152 Amp Gibson.JPG]] 

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 

[[Image:1152 0615.JPG]] 

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

[[Image:1152 Amp 0618.JPG]]

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

[[Image:0618 Gibson.JPG]]

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 

[[Image:0618_Digest.JPG]] 

{| width="200" cellspacing="1" cellpadding="1" border="1"
|-
| 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 ===

[[Image:1152 screening.JPG]]

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

[[Image:1151 1151 Screening.JPG]]

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

[[Image:0621_redigest.JPG]]

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

== '''Result intepretation/Summary''' ==

== '''Conclusion''' ==

[[Category:Cloning]]

1152 pET-Exo-Taq-sso7d - Revision history

Jennifer.truong at 19:22, 21 June 2012