Notebook/September.html

September 1st, 2009

Vectors with His-tag at N-terminus of the construct containing KSI-DP, and KSI-DP-P2/33 were transformed into DH5a cells. Vectors with His-tag at N-terminus of the construct containing ELST and LL37 were cut for front vector and front insert. They will be used to prepare construct KSI-DP_LL37_ELST.

Restrictions from yesterday were put on gel and APH1 (NgoMIV/PstI) was cut out to be purified tomorrow. Control restrictions of Construct no. 29 do not seem to be successful, because the picture is not clear enough. One colony was sent to be sequenced.

Sequencing results showed, that some restriction sites of vector with His-tag on N-term. were changed successfully (MCS-P-ccdB).

We performed control restriction to confirm the presence of GyrB(mod), GyrB-p53, GyrB-CutA1 and GyrB-foldon constructs in vectors with His-tag at N-terminus of the construct. Vector with His-tag at N-terminus of the construct containing GyrB(mod) was cut for back vector.

DH5a cells were transformed with ligations of front vectors P1P3P5 and P6P4P2 with front insert KSI-DP. BL21 cells were transformed with vectors with His-tag at N-terminus of the construct containing foldon-p53, GyrB-p53, GyrB-CutA1 and GyrB-foldon.

nYFP PCR ligaton product was cut, purified and ligated in vector with His-tag at N-terminus of the construct.

September 2nd, 2009

Front vector (ELST) and front insert (LL37) were put on electrophoresis and after isolation front insert was ligated into front vector. DH5a cells were transformed with this construct.

Several colonies (KSI-DP, KSI-DP-P3/22) were inoculated for minipreps.

APH1 (NgoMIV/PstI) fragments were purified from gel. The sequencing results showed, that there four glutamates were not inserted successfully in front of terminator in vector with His-tag on N-term., therefore more clones were tested by control restriction and put on gel electrophoresis.

We ligated vector with p53 inserted with APH1 fragments in order to obtain Construct no. 28 (=vector with His-tag on N-term., containing p53 and APH1, which are bound by 2 amino acid scar).

GyrB(mod) vector was isolated from gel and ligated with CutA1 back insert to obtain GyrB-CutA1 construct with extended linker.

DH5a cells were transformed with GyrB(mod)-CutA1 ligation, newly arrived plasmids APH_BCR and APHE_NZ_CZ and LL-37-ELST ligation.

Several colonies of bacteria transformed with vectors containing KSI-DP-P1P3P5 and KSI-DP-P6P4P2 were inoculated for minipreps.

September 3rd, 2009

Vectors with His-tag at N-terminus of the construct containing KSI-DP and KSI-DP-P2/33 were isolated. Control restriction confirmed the presence of KSI-DP in vector but not the presence of KSI-DP-P2/33. More colonies will be tested. Isolated vector with KSI-DP was send to be sequenced.

Several colonies containing vectors with His-tag at N-terminus of the construct with LL37_ELST, GyrB(mod)-CutA1 and colonies containing APH_BCR and APHE_NZ_CZ were inoculated.

Dh5a cells were transformed with newly arrived vectors containing genes EWAND_TetShak and Vasp_Vero5.

Transformation of Construct no. 28 into DH5a cells.

Ligation of vector, containing foldon and his-tag on N-term with p53 fragment was performed again and transformed into DH5a cells, because control restriction of Construct no. 29 had not been successful.

Preparation of new λ DNA (HindIII/EcoRI) electrophoresis standard ladder.

Several clones of vectors with His-tag at N-terminus of the construct containing KSI-DP-P1P3P5 and KSI-DP-P6P4P2 were isolated from mini-preps. Control restriction was made. One clone of KSI-DP-P1P3P5 and 3 clones of KSI-DP-P6P4P2 were later isolated from remaining content of mini-preps using a kit for plasmid isolation.

DH5a competent cells were prepared.

September 4th, 2009

More colonies containing vectors with KSI-DP-P3/22 were tested and several appropriate were found. One clone was send to de sequenced.

Vectors with His-tag at N-terminus of the construct containing LL37_ELST were isolated from Minipreps and control restriction showed that they contain appropriate construct.

Colonies of bacteria transformed with vectors containing genes EWAND_TetShak and Vasp_Vero5 were inoculated for minipreps.

Control restriction on vectors with His-tag at N-terminus of the construct containing KSI-DP-P1P3P5 and KSI-DP-P6P4P2 was repeated using 4 clones that were isolated with a kit. The results suggested that all clones contained desired constructs. BL21 cells were later transformed with one clone of each construct.

Vectors with His-tag at N-terminus of the construct with GyrB(mod)-CutA1 and vectors APH_BCR and APHE_NZ_CZ. Control restriction was made on clones of vector with His-tag at N-terminus of the construct with GyrB(mod)-CutA1 to confirm the presence of desired construct. Vectors APH_BCR and APHE_NZ_CZ were cut in order to obtain individual inserts, which were later isolated from gel.

Constructs KSI-DP-P1P3P5, KSI-DP-P6P4P2, Gyr(mod)-CutA1 and KSI-DP-P3/22 were sent for sequencing.

September 5th, 2009

Vector with His-tag at N-terminus of the construct containing LL37_ELST was cut with EcoRI and XbaI to prepare front vector in which KSI-DP front insert will be ligated.

Vectors containing genes EWAND_TetShak and Vasp_Vero5 were isolated.

Fragments containing genes for APH, BCR, APHE, NZ and CZ were cut again and purified. APH and BCR were ligated in vector with His-tag at N-terminus of the construct. DH5a cells were transformed with mentioned ligations.

September 6th, 2009

Inoculum with construct 28 and 29.

September 7th, 2009

Manually isolated Constructs 28 and 29 were analyzed using appropriate enzymes, but it was showed, that ligation was not successful. We are going to prepare them in a different way: as frontvectors and frontinserts.

Transformation of vector, containing 4 glutamates in front of terminator into DB3.1 cells.

Vectors APH_BCR and APHE_NZ_CZ were cut again to obtain individual inserts, which were later isolated from gel. Vector with His-tag at N-terminus of the construct was cut so that inserts could be ligated into it.

APHE, NZ and CZ (from September 5th) were ligated in vector with His-tag at N-terminus of the construct.

Mini-preps were inoculated with colonies containing Vector with His-tag at N-terminus of the construct, Vector with His-tag at N-terminus of the construct with foldon, p53, H1P2, P1H1P2, GyrB-foldon and GyrB-p53.

KSI-DP front insert was ligated into vector with His-tag at N-terminus of the construct containing LL37_ELST. DH5a cells were transformed with this construct.KSI-DP front insert was ligated into vector with His-tag at N-terminus of the construct containing LL37_ELST. DH5a cells were transformed with this construct.

Genes EWAND, TetShak, Vasp and Vero5 were cut out of the Mr.Gene vector. Only the restriction of EWAND insert was successful.

September 8th, 2009

Fragments APH, BCR, APHE, CZ and NZ were cut so that they could be ligated in vector with His-tag at N-terminus of the construct and purified. APH and BCR were ligated in vector with His-tag at N-terminus of the construct. DH5a cells were transformed with ligations of APH, BCR, APHE, CZ and NZ in vector with His-tag at N-terminus of the construct.

Control restriction was performed on clones of vector with His-tag at N-terminus of the construct containing H1P2 and P1H1P2 constructs. The results shoved that none of the clones contained desired construct.

Control restriction was also made on clones of vector with His-tag at N-terminus of the construct containing constructs GyrB-foldon and GyrB-p53. None of the tested clones contained mentioned constructs.

Vector with His-tag at N-terminus of the construct containing P1 was cut for front vector so that KSI-DP could be inserted.

Colonies of bacteria, transformed with vector with His-tag at N-terminus of the construct containing KSI-DP_LL37_ELST were inoculated for minipreps.

Isolaton of vector with His-tag on N-term. alone, the one containing foldon and another containing p53. Restrictions: APH1 and p53 as frontvectors; foldon and p53 as frontinserts. Restrictions were put on gel, cut out and purified.

The restriction of TetShak, Vasp and Vero5 was performed again, this time successfully. Fragments were isolated from gel and then cut again with NgoMIV and SpeI. Afterwards fragments were ligated into vector with His-tag at the N-terminus.

We set the ligation of P1, P2 and GCN front inserts with ELST front vector. DH5a cells were transformed with ligation products.

September 9th, 2009

Ligation of yesterday restrictions in order to make Constructs 28 and 29.

Vector with His-tag on N-term., containing EEE and AEA respectively were sent to be sequenced.

Isolation of vectors with His-tag on N-term., containing APH1, p53 and foldon were isolated by GeneJET Plasmid Miniprep Kit.

Inoculum with 4E (=bacteria, containing vector with His-tag on N-term. and four glutamates inserted in front of terminator).

Control restriction of isolated vector with His-tag at N-terminus of the construct containing KSI-DP_LL37_ELST revealed that none of the colonies contain vectors with appropriate construct. Ligation of KSI-DP front insert into vector with His-tag at N-terminus of the construct containing LL37_ELST was set again and ligation product was transformed into DH5a cells.

Vectors with His-tag at N-terminus of the construct containing EWAND, TetShack, VASP and Vero5 were transformed into DH5a cells.

Colonies containing Vectors with His-tag at N-terminus of the construct containing P1_ELST, P2_ELST, GCN_ELST were inoculated for minipreps.

Processed data obtained from AEA CD spectrum: helical structure was confirmed and Tm was calculated using Boltzmann fit - Tm=53oC +/- 1oC.

Vector with His-tag at N-terminus of the construct containing P6 was cut for front vector so that KSI-DP could be inserted.

Vector with His-tag at N-terminus of the construct containing GyrB was cut for front vector and vector with His-tag at N-terminus of the construct containing p53 for front insert. Front vectors from today and yesterday (P1) and front insert were isolated from gel.

Control restriction was performed on clones of vector with His-tag at N-terminus of the construct containing constructs APH,BCR,NZ, APHE and CZ. The bands on gel were on the verge of being invisible, so restriction will be repeated tomorrow.

September 10th, 2009

We isolated some vectors with His-tag on N-term. and 4 inserted glutamates in front of terminator (4E) and performed control restrictions.

Some colonies containing Construct 28 and 29 (ligated as frontvectors+frontinserts) were inoculated in miniprep.

Vectors with His-tag at N-terminus of the construct containing P1_ELST, P2_ELST, GCN_ELST were isolated. Control restriction confirmed the presence of appropriate construct in vectors.

Vector with His-tag at N-terminus of the construct containing ELST_ELST_ELST was isolated. Control restriction confirmed the presence of appropriate construct in vector.

Some colonies containing KSI-DP_LL37_ELST and EWAND, TetShack, VASP, Vero5 were inoculated for miniprep.

Constructs H1-P2, P4-P2, KSI-DP-P1, KSI-DP-P6 and GyrB-P53 were ligated and later transformed in DH5a cells. BL21 cells were transformed with vector with His-tag at N-terminus of the construct containing GyrB-CutA1 with longer linker.

Isolation of vectors with His-tag on N-term., containing APH, BCR, NZ, APHE and CZ were isolated by GeneJET Plasmid Miniprep Kit. 3 clones of vector vith APH were cut for front insert and front vector. The length of front insert fragments suggested that ligation of the construct in vector was successful. Front inserts and front vectors were isolated from gel. Control restriction was made on vectors with BCR, NZ, APHE and CZ. Lengths of the fragments were not as expected. Discussions of what went wrong are still in progress. More clones of vector with BCR, NZ, APHE and CZ were inoculated in mini-preps.

September 11th, 2009

We isolated vectors with His-tag on N-term. containing Construct 28 and 29 and performed restriction analysis, which showed, that constructs are probably successfully inserted.

Control restrictions of 4E from yesterday also showed, that four glutamates are successfully inserted in front of terminator in vector with His-tag on N-term.

We isolated vectors with His-tag on N-term. containing EWAND, Tetshack, VASP, Vero5 and after control restriction we found out that only EWAND and TetShack were successfully ligated into vector. Vector containing TetShack was cut as Front insert to be ligated into APH Front vector.

We isolated vectors with His-teg on N-term. containing KSI-DP_LL37_ELST manualy. Control restriction showed which colony should be isolated using GeneJet Kit.

Two samples were taken to TEM analysis: 1. EWAND: 0,4 ug/mL in 40% MeOH, HEPES 7,5 - 5 mM 2. AEA: 0,4 ug/mL in 5 mM HEPES 7,5 Nothing conclusive has been seen (in AEA sample maybe some linear stacking) - we are waiting for photos to be developed, because they usualy show more detail. The both samples should be analysed with HRTEM (at IJS) for better resolution.

Constructs foldon-APH, APH-p53, p53-APH and APH-P2 were ligated. DH5a cells were transformed with mentioned ligations.

Vectors with His-tag at N-terminus of the construct containing BCR, NZ, APHE and CZ were isolated from mini-preps. After that control restriction was made. The fragments were jet again nearly invisible on gel.

September 12th, 2009

Vectors with His-tag on N-term. containing KSI-DP_LL37_ELST was cut as front insert, vectors with His-tag at N-terminus of the construct containing P1_ELST, P2_ELST, GCN_ELST were cut as front vectors.

TetShack front insert was isolated from gel and ligated into APH front vector.

September 13th, 2009

Inoculation of 4E, H1-P2, P4-P2, KSI-DP-P1, KSI-DP-P6, GyrB-P53, foldon-APH, APH-p53, p53-APH, APH-P2, P2 back, P6 back, p53 back and APH back for miniprep.Inoculation of 4E, H1-P2, P4-P2, KSI-DP-P1, KSI-DP-P6, GyrB-P53, foldon-APH, APH-p53, p53-APH, APH-P2, P2 back, P6 back, p53 back and APH back for miniprep.

September 14th, 2009

4E was isolated. 4E, constructs 28 and 29 were sent to be sequenced. T1W_EEE was transformed into DH5a, because there was not plasmid enough to be sequenced with reverse primer. It will be sequenced again using reverse primer in order to make sure, that the mutation in restriction site for PstI was an artifact of previous sequencing (because it was at the end of sequence). Vector, containing T1W_EEE was cut to acquire frontvector for Construct no. 47.

Restrictions of KSI-DP_LL37_ELST front insert, P1_ELST, P2_ELST, GCN_ELST front vectors were put on gel and front vectors and inserts were isolated. Restriction of P2_ELST was not successful and will be repeated. Front inserts were ligated into front vectors.

Vector with His-tag on N-term. containing KSI-DP_LL37_ELST was send to be sequenced.

We transformated vector with His-tag on N-term. containing KSI-DP_LL37_ELST into BL21 cells and vector with His-tag on N-term. containing TetShack_APH into DH5a cells.

Vectors with His-tag on N-term. containing H1-P2, P4-P2, KSI-DP-P1, KSI-DP-P6, GyrB-P53, foldon-APH, APH-p53, p53-APH and APH-P2 were isolated. Control restriction was made. The results showed that at least one clone of each construct is OK. One clone of each vector was later isolated from using a kit.

September 15th, 2009

Restricted T1W-EEE was put on gel and isolated. RADA primers were annealed and ligated into vector with His-tag on N.term. T1W_EEE and vector with His-tag on N.term. containing KSI-DP were also ligated in order to form Construct no. 47. Inoculum of T1W_EEE for miniprep.

Vectors with His-tag on N-term. containing KSI-DP-P1 and KSI-DP-P6 were cut for front insert; vectors with His-tag on N-term. containing H1-P2, P4-P2 and APH-P2 were cut for front vectors. Fragments were later isolated from gel. Constructs KSI-DP-P1-H1-P2, P1-H1-P2, KSI-DP-P6-P4-P2, P6-P4-P2, GyrB-p53 and P1-APH-P2 were ligated.

Constructs foldon-APH, APH-p53 and p53-APH were sent for sequencing.

Vector with His-tag on N-term. containing P2_ELST was isolated and than cut as front vector which was than isolated from gel. Front insert KSI-DP_LL37_ELST was ligated into front vector.

Few colonies of bacteria transformed with Vector with His-teg on N-term. containing TetShack_APH were inoculated for minipreps.Few colonies of bacteria transformed with Vector with His-teg on N-term. containing TetShack_APH were inoculated in minipreps.

Constructs KSI-DP_LL37_ELST_3xELST, KSI-DP_LL37_ELST_P1_ELST, KSI-DP_LL37_ELST_GCN_ELST were transformed into DH5a cells. Constructs 1xELST, 2xELST, 3xELST were transformed into BL21 cells.

September 16th, 2009

T1W_EEE was isolated in order to be sequenced tomorrow. RADA and T1W_EEE+KSI-DP (in vector with His-tag on N-term.) and mNGF were transformed into DH5a cells.

Manual isolation of construct TetShack_APH was performed. Appropriate colonies was selected and isolated with GeneJet Kit.

Several colonies containing constructs KSI-DP_LL37_ELST_3xELST, KSI-DP_LL37_ELST_P1_ELST, KSI-DP_LL37_ELST_GCN_ELST were inoculated for Miniprep.

DH5a cells were transformed with construct KSI-DP_LL37_ELST_P2_ELST.

DH5a cells were transformed with yesterdays ligations(KSI-DP-P1-H1-P2, P1-H1-P2, KSI-DP-P6-P4-P2, P6-P4-P2, GyrB-p53, P1-APH-P2 and backligations). BL21 cells were transformed with vectors with His-tag on N-term. containing p53-APH, APH-p53, and foldon-APH.

September 17th, 2009

Manual isolation and control restriction of constructs KSI-DP_LL37_ELST_3xELST, KSI-DP_LL37_ELST_P1_ELST, KSI-DP_LL37_ELST_GCN_ELST was performed. Appropriate colonies was selected and isolated with GeneJet Kit. Vectors were sent to be sequenced.

T1W_EEE was sent to be sequenced, this time using reverse primer. Inoculum of mNGF (in original vector) and RADA and Construct 47 in vectors with His-tag on N.term for miniprep.

New stock of BL21 competent cells was made.

4 colonies for each of the constructs KSI-DP-P1-H1-P2, P1-H1-P2, KSI-DP-P6-P4-P2, P6-P4-P2, GyrB-p53, P1-APH-P2 were inoculated for miniprep. One colony of each backligation was also inoculated for miniprep.

The following samples were taken to TEM (MF): GyrCutA1+cum, AEA+Ag, AEA (annealing), sole AEA (two concentrations), DimTetra in TRIS, DimTetra+nanogold and AEA+nanogold.

September 18th, 2009

Plasmid isolations: - RADA in vector with His-tag on N-term. - Construct no. 47 - mNGF restriction analysis of all of them --> ok; mNGF was cut out of gel in order to be ligated into vector with His-tag on N-term.

Manual isolation and control restriction of construct KSI-DP_LL37_ELST_P2_ELST was performed. Appropriate colonies will be isolated with GeneJet Kit.

Constructs KSI-DP_LL37_ELST_3xELST, KSI-DP_LL37_ELST_P1_ELST, KSI-DP_LL37_ELST_GCN_ELST and TetShack_APH (BCR) were send to be sequenced.

DH5a cells were transformed with construct LL37_ELST_GCN_ELST.

Clones of KSI-DP-P1-H1-P2, P1-H1-P2, KSI-DP-P6-P4-P2, P6-P4-P2, GyrB-p53 and, P1-APH-P2 were isolated from mini-preps and control restriction was made. One clone of each construct was later isolated from remaining content of mini-prep using a kit.

• Apparently there was a mix up, result of which is that constructs we thought contained APH, really contain BCR instead.

September 19th, 2009

Vector containing construct KSI-DP_LL37_ELST_P2_ELST was isolated using GeneJet Kit.

Several colonies containing vectors with inserts LL37_ELST_GCN_ELST were inoculated for miniprep. One colony with backligation was also inoculated for miniprep.

Ligation of constructs VASP and Vero5 into vector with His-tag at the N-term. was set.

Vector containing APH_BCR was isolated from mini-preps. Vector was then cut to obtain individual constructs APH and BCR, which were later isolated from gel. Construct mNGF cut with NgoMIV and SpeI was also isolated from gel. APH and BCR were then cut with NgoMIV and SpeI and purified. APH, BCR and mNGF were ligated into vector with His-tag on N-term. of the construct. DH5a cells were transformed with mentioned ligations and vector containing APHE_NZ_CZ.

September 20th, 2009

Several clones of APH, BCR and mNGF in vector with His-tag on N-term. of the construct were inoculated for miniprep.

September 21st, 2009

RADA in vector was restricted and cut out of gel in order to be ligated as front insert into mNGF in vector with His-tag on N-term. Construct no. 47 was sent to be sequenced.

Manual isolation and control restriction of Vector with His-tag on N-term. containing LL37_ELST_GCN_ELST were performed to find out which colony contains vector with appropriate construct. Vector from this colony was than isolated using GeneJet Kit.

Vectors with His-tag on N-term. containing VASP and Vero5 were transformed into DH5a cells. Vector with His-tag on N-term. containing KSI-DP_LL27_ELST_GCN_ELST was transformed into BL21 cells.

Vectors with His-tag on N-term. containing KSI-DP_LL37_ELST_P2_ELST was sent to be sequenced.

Several clones of APH, BCR and mNGF in vector with His-tag on N-term. of the construct were isolated from mini-preps and control restriction was made. Vector with APH was then cut for front vector and front insert and mNGF was cut as front vector.

September 22nd, 2009

RADA as front insert was purified from gel, mNGF was cut again as front vector and purified form gel --> both were ligated.

APH, BCR and mNGF in vector with His-teg on N-term. were isolated from mini-preps. Vector with APH was then cut for front vector and front insert and vector containing mNGF was cut as front vector. Vectors containing constructs ELST, ELST-ELST and LL37_ELST_GCN_ELST were cut as front inserts. All cut vestors and inserts were then isolated from gel. Later, constructs ELST-mNGF, ELST-ELST-mNGF, LL37_ELST_GCN_ELST-mNGF, RADA-mNGF, p53-APH, APH-p53, foldon-APH and APH-P2 were ligated.

17ml of AEA was unfrozen (solution looked clear). Concentration was measured (c=0,05mg/ml) and the sample was concentrated to 0,13 and 0,16 mg/ml. The last was dialysed overnight in 20mM HEPES.

September 23rd, 2009

Vector with His-tag on N-term. and vector APHE_NZ_CZ were isolated from mini-prep. Vector APHE_NZ_CZ was then cut to obtain individual inserts, which were later isolated from gel and cut again with NgoMIV and SpeI.

DH5a cells were transformed with LL37_ELST_GCN_ELST-mNGF, RADA-mNGF, p53-APH, APH-p53, foldon-APH and APH-P2 ligations and vector containing ELST-ELST-ELST.

September 24th, 2009

We took some samples (including gyrCutA1, gyrfoldon, AEA) to DLS, but the visit was mere exploratory - above all we should have had a few times lower concentration of the samples, they should have been filtered and not centrifuged. Method can be also used only to show relative difference and not absolute particle dimensions.

Vectors with His-tag on N-term. containing VASP and Vero5 were isolated. Control restriction confirmed the presence of inserts of appropriate lenght.

Vectors containing EWAND and TetShack were once again isolated.

Vector containing ELST-ELST-ELST was isolated from mini-prep and cut as front insert. There were no visible fragments (front insert) on gel, so restriction will have to be repeated.

APHE, NZ and CZ fragments cut with NgoMIV and SpeI were purified and ligated in vector with His-tag on N-term. DH5a cells were transformed with these ligations.

In the morning several clones of bacteria containing vectors with RADA-mNGF and LL37-ELST-GCN-ELST-mNGF were inoculated for miniprep. Vectors were later isolated from mini-preps and control restriction was made. One flask was then inoculated with bacteria containing vector with RADA-mNGF construct.

September 25th, 2009

Constructs EWAND and TetShack were cut out of the vector, put on gel and isolated from it.

Several clones of vectors that supposedly contained constructs APH-p53, p53-APH, foldon-APH and APH-P2 were isolated from mini-preps and control restriction was made. One clone of vector for each construct was later isolated from remaining content of mini-preps using a kit.

Vectors containing ELST-ELST-ELST and RADA-mNGF were isolated from mini-preps. Vector containing ELST-ELST-ELST was then cut as front insert.

10 mL media were inoculated with 3 colonies containing vectors with APHE, NZ or CZ for miniprep.

September 26th, 2009

Restriction mix of vector containing ELST-ELST-ELST was put on gel. Front insert was then isolated from gel.Constructs LL37-ELST-GCN-ELST-mNGF and ELST-ELST-ELST-mNGF were ligated. DH5a cells were transformed with mentioned ligations.

Several clones of vectors containing APHE, NZ or CZ were isolated from mini-preps and control restriction was made. The results showed that all clones were OK.

September 28th, 2009

Vector with P1 was cut for front insert and vector containing APH-P2 was cut for front insert. Fragments were then isolated from gel and constructs P1-H1-P2 and P1-APH-P2 were ligated. DH5a cells were transformed with ligations.

Quick change site directed mutagenesis was performed on all constructs containing p53 in order to lose a PstI restriction site in the constructs.

September 29th, 2009

Control restriction was made on several clones of vector supposedly containing construct ELST-ELST-ELST-mNGF.

September 30th, 2009

Control restriction of vector with His-tag on N-term. containing KSI-DP_P2/33_LL37_ELST_GCN_ELST confirmed the presence of this construct in vector.

Control restriction was made on several clones of vector supposedly containing constructs P1-H1-P2 or P1-APH-P2. One clone of each construct was then isolated from mini-prep.

Victor containing construct LL37-ELST-GCN-ELST was cut for front insert, fragment was isolated from gel and construct LL37-ELST-GCN-ELST-mNGF was ligated.