Xiaojing.Wang at 19:41, 19 July 2012

2012-07-19T19:41:07Z

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

'''Purpose:''' Cloning of p8H-VentExo 

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

'''Notebook: '''

'''Page:'''

'''Clone ID: '''1156

'''Experimenters: '''Jennifer Truong

== '''Results''' ==

=== Cloning ===

Date: June 13, 2012 

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

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

[[Image:0613 Amplifacation.JPG]] 

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

[[Image:0613 Reamp Digestion.JPG]]

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

Date: June, 14 2012

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

Date: June 22, 2012 

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

[[Image:1248 Amp.JPG]] 

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

[[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. 

Date: July 10, 2012 

[[Image:0710 amp.JPG]] 

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

[[Image:0710 digestion.JPG]] 

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

Date: July 11, 2012 

[[Image:1156 amp.JPG]] 

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

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

[[Image:1156 digestion.JPG]]

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

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

=== Colony Screening ===

Date: 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 media overnight

Date: 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. 

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

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

{| cellspacing="1" cellpadding="1" border="1" width="239" style=""
|-
| Lane 
| Sample 
| Enzymes 
|-
| 1 
| 1 
| Xho1 + Kpn1 
|-
| 2 
| 1 
| Xho1 + Kpn1 
|-
| 3 
| 2 
| HindIII + Xho1 
|-
| 4 
| 2 
| HindIII + Xho1 
|}

Submitted sample one for sequencing 

Date: 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 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: July 16, 2012 

[[Image:1156 0716 Screening.JPG]] 

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

Date: July 17, 2012 

[[Image:0717 Digestion.JPG]] 

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

Date: July 19, 2012 

[[Image:1156_0719_Screening.JPG]] 

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

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

== '''Conclusion''' ==

 

[[Category:Cloning]]

1156 p8H-VentExo - Revision history

Xiaojing.Wang at 19:41, 19 July 2012