Revision as of 17:05, 14 September 2016

Wednesday 14^th September

Lab work

Visualization

Glycerol stocks of clones 2, 7, 8 and 12 containing GFP 1.9 in pSB1C3

"By Maxence & Mahnaz"

The glycerol stock of the bacteria with the following plasmids were made.

• pPS16_009 (GFP 1.9) clone 2
• pPS16_009 (GFP 1.9) clone 7
• pPS16_014 (GFP 1.9) clone 8
• pPS16_014 (GFP 1.9) clone 12

500µL of liquid culture and 250 µL of glycerol 60% were mixed and put at -20°C.

Plasmids extraction of clones 2, 7, 8 and 12 containing GFP 1.9 in pSB1C3

"By Maxence & Mahnaz"

The following plasmids were extracted using the "Charge Switch-Pro Plasmid Miniprep" kit:

• pPS16_009 (GFP 1.9) clone 2
• pPS16_009 (GFP 1.9) clone 7
• pPS16_014 (GFP 1.9) clone 8
• pPS16_014 (GFP 1.9) clone 12

Samples preparation for sequencing

By Maxence & Mahnaz

20 µL of plasmids pSB1C3 GFP 1.9 (clones 2, 7, 8 and 12) were sent to be sequenced. 20 µL of the primers iPS84 (5µM) and iPS140 (5µM) were sent for sequencing.

NanoDrop Measurements

By Maxence & Mahnaz

PCR of gblock 1.1 - 1.2 (fragment 1) and gblock 2.1 - 2.2 (fragment 2) Ligation products

By Maxence & Mahnaz

With the results obtained the 12th and 13th September, a new amplification approach was tested: the Ligation products from the 12th were diluated at 1/10 and two buffer (HF and GC) were tested with others different PCR conditions. Furthermore, ligations products from the 13th September were also amplified.

For each 50μl of reaction, mix the following reagents :

• 1 µL of matrix
• 1 µL of dNTPs (10mM)
• 2.5 µL of each primer mix (10µM)
• 10 µL of buffer (5X)
• 0,5 µL of Phusion polymerase
• 32.5 µL of nuclease free water

Mix gently and aliquot 50 μl of the mix into the PCR tubes on ice. Perform PCR as follow:

Primers used were:

Gel of gblock 1.1 - 1.2 (fragment 1) and gblock 2.1 - 2.2 (fragment 2) Ligation products

By Maxence & Mahnaz

3 µL of each PCR products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.

PCR products expected were :

GEL bizare

The results were the same as previous ones.

Another strategy was to make a PCR directly of fragments 1 and 2 obtained previously even if these fragments were not working for Gibson. Fragments 1 and 2 from the 2nd September were amplified with the same protocol as mentioned above.

GEL

PCR of gblock detection, gblock ST sgRNA, gblock NM sgRNA, gblock spacer and plasmid PZA11

By Maxence & Mahnaz

In order to obtain pZA11 containing the desired sequences, gblocks were amplified in order to run Gibson. For that purpose, PCR was performed with the following protocol.

For each 50μl of reaction, mix the following reagents :

• 1 µL of matrix
• 1 µL of dNTPs (10mM)
• 2.5 µL of each primer mix (10µM)
• 10 µL of buffer HF (5X)
• 0,5 µL of Phusion polymerase
• 32.5 µL of nuclease free water

Mix gently and aliquot 50 μl of the mix into the PCR tubes on ice. Perform PCR as follow:

Primers used were:

PCR Clean-up of PCR products

By Maxence & Mahnaz

PCR products obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit protocol.

Gel of cleaned up PCR products

By Maxence

After amplification, 3 µL of each cleaned up PCR products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.

PCR products expected were :

GEL ?

All PCR products were at the good size but there were no results obtained for NM sgRNA.

Another PCR was run in order to obtain PCR products from NM sgRNA, the good TM was used (63°C).

GEL

Linearization of PCR product pZA11

By Maxence & Mahnaz

PCR product PZA11 has been linearized for further Gibson application by using DpnI treatment :

• 30 µL of cleaned up PCR product GFP 11 - pSB1C3
• 4 µL of fast digest buffer
• 1 µL of DpnI
• 5 µL of water

The mix was placed 10 minutes at 37°C and was cleaned by using the NucleoSpin Gel and PCR Clean-up kit protocol.

PCR of pSB1C3 from dCas9 ST - GFP 11 clone 8

By Maxence & Mahnaz

In order to obtain FKBP - GFP 10 in pSB1C3 by Gibson, pSB1C3 must be amplified in order to run Gibson. For that purpose, PCR was performed with the following protocol.

For each 50μl of reaction, mix the following reagents :

• 1 µL of matrix
• 1 µL of dNTPs (10mM)
• 2.5 µL of each primer mix (10µM)
• 10 µL of buffer HF (5X)
• 0,5 µL of Phusion polymerase
• 32.5 µL of nuclease free water

Mix gently and aliquot 50 μl of the mix into the PCR tubes on ice. Perform PCR as follow:

Primers used were:

PCR Clean-up of PCR products pSB1C3

By Maxence & Mahnaz

PCR products obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit protocol.

Gel of cleaned up PCR products pSB1C3

By Maxence & Mahnaz

After amplification, 3 µL of each cleaned up PCR products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.

PCR products expected were :

GEL ?

PCR products were at the good size.