Editing 1156 p8H-VentExo

== '''Cloning of p8H-VentExo'''  ==

'''Purpose:''' Cloning of p8H-VentExo<br> 

'''Date: '''2012-06-13 

'''Notebook:&nbsp;''' 

'''Page:''' 

'''Clone ID: '''1156 

'''Experimenters: '''Jennifer Truong 

== '''Results'''  ==

=== Cloning  ===

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

Ran PCR using 423 pET30b-DeepVent-sso7d as template and VentFire-Nde1-F and VentFire-Xho1-R as primers. Used KAPA&nbsp;enzyme with 20 cycles and 45 seconds extension time. <br> 

Ran PCR&nbsp;gel, product is supposed to be ~1.3 kb, see lanes 9 and 10 

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

Cut out bands (lane 9 and 10) , purified DNA and digested DNA and vector p8H&nbsp;(1135)&nbsp;with restriction enzymes Xho1 and Nde1 

[[Image:0613 Reamp Digestion.JPG]] 

Lane 6 is the DNA&nbsp;digestion and lane 7 is the p8H vector digestion. Cut out bands, purify and ligate overnight. 

Date:&nbsp;June, 14 2012 

Tranformed cells with DH5 alpha cells in the morning, plated on KAN plates 

Date: June 22, 2012<br> 

Cells came with with a mutation, might have had a tail to tail join. Started all over<br> 

[[Image:1248 Amp.JPG]]<br> 

Cut out band in lane 1 (expected size 1,365 bp) , digested with p8H (1135)&nbsp;with restriction enzymes Nde1 and Xho1<br> 

[[Image:0622 Digestion.JPG]] 

Cut out bands in lanes 2 and 3, prepared for ligation 

May have accidentally switched samples, reamplified sequence 

[[Image:0622 Amp.JPG]] 

No band in lane 1, need to reamplify on Monday, expected size is 1.4 kb. <br> 

Date:&nbsp;July 10, 2012<br> 

[[Image:0710 amp.JPG]]<br> 

Cut out band in lane 1 (expected size 1,365 bp) , digested with p8H (1135) with restriction enzymes Nde1 and Xho1<br> 

[[Image:0710 digestion.JPG]]<br> 

Digestion of DNA did not appear (lane 2), saved digested vector (lane 2), reamplify and digest tomorrow<br> 

Date:&nbsp;July 11, 2012<br> 

[[Image:1156 amp.JPG]]<br> 

Reamplified DNA from 423 pET30b-DeepVent-sso7d as template and VentFire-Nde1-F and VentFire-Xho1-R as primers, used PHUSION&nbsp;enzyme 25 cycles (see lanes 1 and 2)<br> 

Cut out bands, purified and digested with restriction enzymes Nde1 and Xho1<br> 

[[Image:1156 digestion.JPG]] 

Digested DNA with Nde1 and Xho1, ligated it together with digested vector&nbsp; p8H (1135) from yesterday 

Ligate over night, transform with Top10 cells and plate on KAN plates tomorrow morning 

=== Colony Screening  ===

Date:&nbsp;June 15, 2012 

Colonies grew on plates, screened 7 samples for PCR screening 

[[Image:1156 PCR Screening.JPG]] 

All samples positive, grew 1, 2, 4, 5, 6 and 7 is KAN&nbsp;media overnight 

Date:&nbsp;June 18, 2012 

Did Miniprep, purified plasmids, digested with Nde1 and Xho1 to confirm positive clones 

[[Image:1156 Digestion 0618.JPG]] 

Size of band is a little too big. Digested with different enzymes to check for fragment sizes. <br> 

DNA ladder 5000, 3000, 2000, '''1500''', 1000, 750, '''500''', 300, 100<br><br> 

[[Image:1156 Digestion 0618 2.JPG]] 

{| cellspacing="1" cellpadding="1" border="1" width="239" style=""
|-
| Lane<br> 
| Sample<br> 
| Enzymes<br>
|-
| 1<br> 
| 1<br> 
| Xho1 +&nbsp;Kpn1<br>
|-
| 2<br> 
| 1<br> 
| Xho1 + Kpn1<br>
|-
| 3<br> 
| 2<br> 
| HindIII&nbsp;+&nbsp;Xho1<br>
|-
| 4<br> 
| 2<br> 
| HindIII + Xho1<br>
|}

Submitted sample one for sequencing <br> 

Date:&nbsp;June 21, 2012 

Sequencing came back with tail to tail, redigest sample 1 to check for the length of the plasmid 

[[Image:0621 redigest.JPG]] 

Redigested 1156 to check for size. Looks like it is a tail to tail join 

Digested 5 uL&nbsp;of sample 2 with HindIII and Kpn1 separately to check 

[[Image:1156 0621 Digest.JPG]] 

{| cellspacing="1" cellpadding="1" border="1" style="width: 235px; height: 92px;"
|-
| Lane 
| Sample 
| Enzyme
|-
| 1 
| 2 
| HindIII
|-
| 2 
| 2 
| KpnI
|}

Date:&nbsp;July 16, 2012<br> 

[[Image:1156 0716 Screening.JPG]]<br> 

Digested plasmids with Nde1 and Xho1, expected size 1.3 kb. All samples postive, sent sample 1 to sequencing (lane 1)<br> 

Date: July 17, 2012<br> 

[[Image:0717 Digestion.JPG]]<br> 

Band is too high (see lane 2) Digested plasmid 1 with Nde1 and Xho1, expected size 1.3 kb<br> 

Date:&nbsp;July 19, 2012<br>

[[Image:1156_0719_Screening.JPG]]<br>

Digested plasmids with Nde1 and Xho1, submitted sample 1 to sequencing<br>

== '''Result intepretation/Summary'''<br>  ==

== '''Conclusion'''  ==

<br> 

[[Category:Cloning]]