1156 p8H-VentExo

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

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

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

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

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

May have accidentally switched samples, reamplified sequence

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

Date: July 10, 2012

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

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

Date: July 11, 2012

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

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

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

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

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

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

Lane	Sample	Enzyme
1	2	HindIII
2	2	KpnI

Date: July 16, 2012

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

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

Date: July 19, 2012

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